Method for activating t cells for cancer treatment

ABSTRACT

The present invention relates to a cancer-specific tumor antigen neoepitope represented by any one of SEQ ID NOs: 1 to 184, an antigen-presenting cell loaded with the neoepitope, and a method for activating T cells for cancer treatment using the antigen-presenting cell. An antigen-presenting cell, that is, a dendritic cell, loaded with a cancer-specific tumor antigen epitope provided in the present invention enables rapid and effective induction of differentiation and proliferation of cancer antigen-specific T cells, preferably memory T cells, and the memory T cells thus activated can treat a cancerous or neoplastic condition or prevent recurrence, progression, or metastasis of cancer while avoiding the defense mechanism of cancer cells.

TECHNICAL FIELD

The present invention relates to a cancer-specific tumor antigenneoepitope, an antigen-presenting cell loaded with the neoepitope, and amethod for activating T cells for cancer treatment using theantigen-presenting cell.

BACKGROUND ART

Gastric cancer is known as a malignancy with a high incidence in theworld, especially in Asia. There have been many known causes ofdevelopment of gastric cancer; however, gastric cancer may be typicallyclassified into EBV-associated gastric cancer, which is caused byinfection with Epstein-Barr virus (EBV), and gastric cancer cellantigen-associated gastric cancer, which is caused by accumulation ofgenetic mutations in gastrointestinal cells. For current treatment forgastric cancer, excision of cancerous tissue has long been known to bethe most effective, and chemotherapy and radiation therapy are alsoperformed. However, it appears that gastric cancer is a hard-to-curedisease when not found early. In addition, although clinical trials havebeen conducted through several biological agents (antibodies, smallmolecules), therapeutic agents with good clinical effects have not yetbeen reported.

Recently, cancer cell-specific targeted therapy using patient-derivedautologous T cells has been studied in several institutions, andclinical trials have been conducted for lymphoma using chimeric antigenreceptor (CAR) T cells in several institutions. As a result, due to goodclinical effects and low side effects, such therapy has attracted muchattention as a new field of anticancer therapy.

Use of patient-derived T cells decreases induction of immune responseswhich is the biggest side effect of cell therapeutic agents, and removesrestrictions on the donor's HLA type. Thus, such T cells have been knownas therapeutic agents which are effective and have no side effects. Todate, CD8+ T cells, CD4+ T cells, NK cells, dendritic cells, and CAR Tcells are known as types of cell therapeutic agents which are mostwidely used in the field of anticancer therapy. NK cells havecell-killing efficacy, and have several side effects due to not havingantigen specificity. Dendritic cells are therapeutic agents belonging tothe vaccine concept in that they have no function of directly killingcells, and are capable of delivering antigen specificity to T cells inthe patient's body so that cancer cell specificity is imparted to Tcells with high efficiency. In addition, CD4+ T cells play a role inhelping other cells through antigen specificity, and CD8+ T cells areknown to have the best antigen specificity and cell-killing effect.

However, most cell therapeutic agents, which have been used or developedto date, have limitations and thus have no clinical effect. Taking alook at the limitations, cancer cells, on their own, secrete substancesthat suppress immune responses in the human body, or do not presentantigens necessary for production of antibodies against such cancercells, thereby preventing an appropriate immune response from occurring.

Meanwhile, dendritic cells not only act as surveillants to detectantigens that come from the outside of the human body or are producedinternally, but also quickly travel to the secondary lymphoid organswith such recognized and absorbed antigen, thereby acting as specializedantigen-presenting cells that present the antigens to immune cells,including T cells, which react with the antigens. Anti-cancerimmunotherapeutic vaccines using dendritic cells have been developedthrough several methods, and may be largely divided into ex vivogenerated dendritic cell vaccines and in vivo dendritic cell vaccines.The in vivo dendritic cell vaccine works in a manner of directlydelivering an antigen to dendritic cells present in the body. Inaddition, a method using the ex vivo generated dendritic cell vaccine isin such a manner that dendritic cells are isolated from the patient'sPBMCs and an antigen to be presented is delivered to the isolateddendritic cells, through which the dendritic cells are activated andthen injected back into the patient so that the antigen is deliveredfrom the injected dendritic cells to T cells. In the latter, ex vivodendritic cell culture method and antigen delivery method are important,and currently used antigen presentation methods include transfection ofDNA of an antigen to be presented using virus or nucleofection, orantigen delivery targeting dendritic cells in which an antigen is boundto an antibody targeting the dendritic cells.

Currently, the biggest problems in dendritic cell vaccines are thatsevere chronic inflammatory phenomena in the body and the Warburg effectare exhibited, and it is considered very difficult to achieve effectiveactivation of anticancer immune cells under the cancer microenvironmentin which immunosuppressive cytokines, immunosuppressive T cells,dendritic cells, and the like are present.

Technical Problem

An object of the present invention is to provide an Epstein-Barr virus(EBV)-positive cancer-specific tumor antigen neoepitope, and acomposition for activating T cells which comprises the same.

Another object of the present invention is to provide anantigen-presenting cell loaded with a neoepitope of the presentinvention, the antigen-presenting cell being capable of activating Tcells for cancer treatment.

Yet another object of the present invention is to provide a T cell,activated by the antigen-presenting cell loaded with a neoepitope of thepresent invention.

Still yet another object of the present invention is to provide a methodfor activating T cells for cancer treatment.

However, the technical problem to be solved by the present invention isnot limited to the above-mentioned problems, and other problems whichare not mentioned will be clearly understood by those skilled in the artfrom the following description.

Solution to Problem

According to an embodiment of the present invention, there is provided acancer-specific tumor antigen epitope.

In the present invention, the “cancer-specific tumor antigen epitope” isderived from a protein antigen which is present only in cancer cells andis not present in normal cells. In the present invention, thecancer-specific tumor antigen epitope includes at least one epitoperecognized by T cell receptors; and such an epitope may preferablyinclude epitopes present in Epstein-Barr virus (EBV)-positive cancercells, including the EBV viral epitopes or cancer cell epitopes, and maymore preferably be the Epstein-Barr virus (EBV)-positive cancer cellantigen which is Epstein-Barr virus latent membrane protein 2 (LMP2a),or Epstein-Barr nuclear antigen 1 (EBNA-1), or a neoepitope thereof.

In the present invention, the “Epstein-Barr virus latent membraneprotein 2 (LMP2a)” is one of several EBV genes expressed in all of typeII and III diseases/malignancies. LMP2a corresponds to a transmembraneprotein that acts as a negative modulator of B cell-receptor signalingand promotes cell survival through sequestering of tyrosine kinases.HLA-A2-restricted peptides are epitope-specific cytotoxic T lymphocytes,which are detectable ex vivo in 60% to 75% of individuals, and are themost immunodominant LMP epitopes in latent diseases. The CLGGLLTMVpeptide has long been seen to be a potential target for NPC and HLtreatments, since the epitope is conserved in biopsies taken from NPCand HL patients and, along with other EBV latent epitopes, isimmunologically weak.

In the present invention, the “Epstein-Barr nuclear antigen 1 (EBNA-1)”corresponds to a multifunctional, dimeric viral protein associated withEpstein-Barr virus. This corresponds to an EBV protein found only in allEBV-associated malignancies. This plays an important role in maintainingthe altered state that cells take when the cells are infected with EBV.EBNA-1, on the other hand, corresponds to a glycine-alanine repeatsequence that separates a protein into amino- and carboxy-terminaldomains. This sequence serves to stabilize a protein, preventproteasomal breakdown, and impair antigen processing and MHC classI-restricted antigen presentation. Thus, the EBNA-1 inhibitsCD8-restricted cytotoxic T cell responses against virus-infected cells.The EBNA-1 is expressed by Qp promoter in all latency programs andcorresponds to the only viral protein expressed in latency program I.

In the present invention, the “neoepitope” refers to an epitope that isnot present in a reference such as normal, non-cancerous cells orgermline cells and is found in cancer cells.

In the present invention, the neoepitope may exhibit binding affinitywith at least one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G,β2-microglobulin, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1,HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOBloci so that T cells, preferably memory T cells, extracted from humanblood can have efficacy. Among these, the neoepitope may include thoseexhibiting high binding affinity with at least one of the HLA types thatare most expressed by Koreans, for example, HLA-A*2402, HLA-A*A0201,HLA-A*3303, HLA-A*1101, HLA-A*0206, HLA-A*3101, HLA-B*5101, HLA-B*4403,HLA-B*5401, HLA-B*5801, and HLA-B*3501.

Preferably, in the present invention, the neoepitope has high bindingaffinity for HLA-A*2402 and may include neoepitopes of LMP2a antigenwhich is a peptide represented by any one of SEQ ID NOs: 1 to 151; orhas high binding affinity for HLA-A*3101 and may include neoepitopes ofEBNA-1 antigen which is a peptide represented by any one of SEQ ID NOs:152 to 184.

Here, in the present invention, for a method of measuring neoepitope-HLAaffinity, NetMHC 3.4 (URL: www.cbs.dtu.dk/services/NetMHC-3.4/) may beused to predict whether a neoepitope binds to a specific HLA allele.However, the present invention is not limited thereto.

In the present invention, the “HLA” or “human leukocyte antigen” refersto human gene that encodes a major histocompatibility complex (MHC)protein on the surface of cells that are responsible for regulation ofthe immune system. “HLA-I” or “HLA class I” refers to human MHC class Igene including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, andβ2-microglobulin loci. “HLA-II” or “HLA class II” refers to human MHCclass II gene including HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1,HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, andHLA-DOB loci.

In the present invention, the cancer may be Epstein-Barr virus(EBV)-positive cancer, including EBV-positive gastric cancer,EBV-positive cervical cancer, EBV-positive Burkitt's lymphoma,EBV-positive T cell lymphoma, EBV-positive breast cancer, EBV-positiveleiomyosarcoma, EBV-positive smooth muscle tumor, EBV-positive Hodgkinlymphoma, EBV-positive nasopharyngeal cancer, or EBV-positivepost-transplant lymphoproliferative disorder (PTLD), with EBV-positivegastric cancer being preferred.

According to another embodiment of the present invention, there isprovided a nucleic acid molecule, encoding a cancer-specific tumorantigen epitope provided in the present invention, preferably aneoepitope of LMP2a or EBNA-1 which is an antigen of Epstein-Barr virus(EBV)-positive cancer cells.

The nucleic acid molecule of the present invention includes any nucleicacid molecule obtained by converting an amino acid sequence of apolypeptide provided in the present invention into a polynucleotidesequence as known to those skilled in the art. Thus, variouspolynucleotide sequences may be prepared due to open reading frame(ORF), all of which are also included in the nucleic acid molecule ofthe present invention.

According to yet another embodiment of the present invention, there isprovided an expression vector, into which the isolated nucleic acidmolecule provided in the present invention is inserted.

In the present invention, the “vector” is a nucleic acid molecule whichis capable of transporting another nucleic acid linked thereto. One typeof vector is a “plasmid,” which refers to circular double-stranded DNAinto which an additional DNA segment can be ligated. Another type ofvector is a phage vector. Yet another type of vector is a viral vector,where an additional DNA segment can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (for example, bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (for example, non-episomal mammalian vectors) can be integratedinto the genome of a host cell upon introduction into the host cell, andthus are replicated along with the host genome. In addition, certainvectors are capable of directing expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” or simply “expression vectors.” In general,expression vectors useful in recombinant DNA techniques are often in theform of plasmids. In the present specification, “plasmid” and “vector”may be used interchangeably as the plasmid is the most commonly usedform among vectors.

Specific examples of the expression vector in the present invention maybe selected from, but are not limited to, the group consisting ofcommercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Tivectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ,T-even, T2, T3, T7; plant viruses. Any expression vector known, to thoseskilled in the art, as expression vectors can be used in the presentinvention, and the expression vector is selected depending on the natureof the target host cell. Introduction of a vector into a host cell maybe performed by calcium phosphate transfection, viral infection,DEAE-dextran-mediated transfection, lipofectamine transfection, orelectroporation. However, the present invention is not limited thereto,and those skilled in the art may adopt and use an introduction methodappropriate for the expression vector and the host cell which are used.The vector may preferably contain at least one selection marker.However, the present invention is not limited thereto, and selection canbe made using the vector that contains no selection marker, depending onwhether or not a product is produced. The selection marker is selecteddepending on the target host cell, which is done using methods alreadyknown to those skilled in the art, and thus the present invention has nolimitation thereon.

In order to facilitate purification of the nucleic acid molecule of thepresent invention, a tag sequence may be inserted into and fused to anexpression vector. The tag includes, but is not limited to,hexa-histidine tag, hemagglutinin tag, myc tag, or flag tag, and any tagknown to those skilled in the art which facilitates purification can beused in the present invention.

According to still yet another embodiment of the present invention,there is provided a host cell, transfected with the expression vectorprovided in the present invention.

In the present invention, the “host cell” includes individual cells orcell cultures which may be or have been recipients of the vector(s) forincorporation of a polypeptide insert. The host cell includes progeny ofa single host cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic DNA complement) to the originalparent cell due to natural, accidental, or intentional mutation. Thehost cell includes cells transfected in vivo with the polynucleotide(s)herein.

In the present invention, the host cell may include cells of mammalian,plant, insect, fungal, or cellular origin, and may be, for example,bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells such as yeast cells and Pichia pastoris; insect cells suchas Drosophila and Spodoptera Sf9 cells; animal cells such as Chinesehamster ovary (CHO) cells, SP2/0 (mouse myeloma), human lymphoblastoid,COS, NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, babyhamster kidney (BHK) cells, human embryonic kidney (HEK) cells, orPERC.6 (human retinal cells); or plant cells. However, the host cell isnot limited thereto, and any cell known to those skilled in the artwhich can be used as a host cell is available.

According to still yet another embodiment of the present invention,there is provided a composition for activating T cells, comprising acancer-specific tumor antigen epitope provided in the present invention,a nucleic acid molecule encoding the same, an expression vector intowhich the nucleic acid molecule is inserted, or a host cell transformedwith the expression vector.

As used herein, the term “activation of T cells” refers to a populationof monoclonal (for example, encoding the same TCR) or polyclonal (forexample, having clones encoding different TCRs) T cells that have T cellreceptors recognizing at least one tumor antigen peptide. Activated Tcells may include one or more subtypes of T cells, including, but notlimited to, one or more selected from the group consisting of cytotoxicT cells, helper T cells, natural killer T cells, γδT cells, regulatory Tcells, and memory T cells, with memory T cells being preferred.

In the present invention, the activated T cells can treat a cancerous orneoplastic condition or prevent recurrence, progression, or metastasisof cancer while avoiding the defense mechanism of cancer cells.

According to still yet another embodiment of the present invention,there may be provided an antigen-presenting cell (APC) loaded with acancer-specific tumor antigen epitope provided in the present invention.

In the present invention, the antigen-presenting cells may include atleast one of dendritic cell (DC), B cell, and macrophage, with dendriticcell being preferred.

In the present invention, the “dendritic cell” refers to any member of adiverse population of morphologically similar cell types found inlymphoid or non-lymphoid tissues. These cells are characterized by theirdistinctive morphology and high expression levels of surface class I andclass II MHC molecules, which are proteins that present antigenicpeptides to T cells. DCs, other APCs, and T cells may be isolated orderived (such as differentiated) from a number of tissue sources, andconveniently from peripheral blood, such as peripheral blood mononuclearcells (PBMCs) derived from peripheral blood.

In the present invention, the antigen-presenting cell can inducedifferentiation and proliferation of cancer antigen-specific T cells,preferably memory T cells, thereby treating a cancerous or neoplasticcondition or preventing recurrence, progression, or metastasis of cancerwhile avoiding the defense mechanism of cancer cells.

According to still yet another embodiment of the present invention,there is provided a fusion protein, comprising: a cancer-specific tumorantigen epitope provided in the present invention; and a dendriticcell-specific antibody or a fragment thereof.

The fusion protein provided in the present invention enables thecancer-specific tumor antigen epitope provided in the present inventionto be loaded on dendritic cells.

In the present invention, the dendritic cell-specific antibody mayinclude, but is not limited to, antibodies specific for DCIR, MHC classI, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19,CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83,CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO,DEC-205, Clec9A, 33D1, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2,IFN-γ receptor, IL-2 receptor, ICAM-1, Fcγ receptor, LOX-1, or ASPGR,which is on dendritic cells.

The cancer-specific tumor antigen epitope in the fusion protein of thepresent invention may be conjugated to the dendritic cell-specificantibody or a fragment thereof. Here, the term “conjugate” refers to anymaterial formed by joining two parts together. A representativeconjugate according to the present invention includes those formed byjoining an antigen together with an antibody and a TLR agonist. The term“conjugation” refers to a process of forming a conjugate and generallyindicates physical coupling, for example, covalent bond, co-coordinatecovalent bond, or second binding force, for example, Van der Waalsbinding force. The process of linking the antigen to the antibody mayalso be done via a non-covalent association such as a dockerin-cohesinassociation (as described in U.S. Patent Publication No. 20100135994,Banchereau et al. relevant portions incorporated herein by reference) orvia a direct chemical linkage by forming a peptide or chemical bond.

According to another embodiment of the present invention, there isprovided a method for producing an antigen-presenting cell, in which theantigen-presenting cell is loaded with a cancer-specific tumor antigenepitope provided in the present invention.

In the present invention, the antigen-presenting cell may include one ormore of dendritic cell, B cell, and macrophage, with dendritic cellbeing preferred.

In the present invention, the dendritic cells (such as immaturedendritic cells) may be obtained from a variety of sources includingautologous sources, that is, derived from a target individual. Thedendritic cells may preferably be obtained from peripheral bloodmononuclear cells (PBMCs) derived from peripheral blood, and morepreferably be obtained by isolating monocytes from individual-derivedPBMCs and contacting the monocytes with a plurality of cytokines. Here,the type of cytokine that induces differentiation of the monocytes intodendritic cells is not particularly limited, and may include, forexample, one or more of GM-CSF and IL-4.

In the present invention, the “target individual” means an individualwho has or is at high risk of developing cancer.

In the present invention, once antigen-presenting cells are prepared asdescribed above, the antigen-presenting cells may be loaded with acancer-specific tumor antigen epitope of the present invention. Ingeneral, immature dendritic cells capture an antigen throughphagocytosis or receptor-mediated endocytosis, process the antigenthrough a series of intracellular processes and then cause an antigenicpeptide to be loaded on MHC and presented to T lymphocytes. With theprocess of processing an antigen, the dendritic cells become moremature, which makes them lose receptors used for phagocytosis andendocytosis, exhibit increased expression of MHC class I, II,costimulatory molecules, and adhesion molecules, and express newchemokine receptors. This allows the dendritic cells to migrate to Tlymphocyte-rich areas of the surrounding lymph nodes, and to present theantigen to T lymphocytes, thereby causing a T lymphocyte immuneresponse.

In an example of the present invention, in order for the cancer-specifictumor antigen epitope to be loaded on the antigen-presenting cell, theantigen-presenting cell may be contacted with the cancer-specific tumorantigen epitope of the present invention, and preferably, a step ofpulsing, with the cancer-specific tumor antigen epitope of the presentinvention, the antigen-presenting cells, for example, immature dendriticcells, or antigen-presenting cells (such as dendritic cells) containedin or derived (for example, differentiated) from PBMCs may be performed.As known in the art, pulsing refers to a process of mixing cells, suchas dendritic cells, with a solution containing a cancer-specific tumorantigen epitope peptide of the present invention, and then optionallyremoving the cancer-specific tumor antigen epitope peptide from themixture. In the present invention, when the immature dendritic cells arecontacted with the cancer-specific tumor antigen epitope, treatment withtoll-like receptor agonists may be performed to further inducematuration of a population of immature dendritic cells. Here, exemplaryTLR agonists include, but are not limited to, polylC, MALP, and R848.

In another example of the present invention, in order for thecancer-specific tumor antigen epitope to be loaded on theantigen-presenting cell, it is possible to perform nucleofection of theantigen-presenting cell with an expression vector, preferably a plasmid,into which a nucleic acid molecule encoding the cancer-specific tumorantigen epitope is inserted. Here, the nucleofection may be performed byany useful means in the art, including, for example, Amaxa®nucleofection system or InVitrogen® nucleofection system.

In yet another example of the present invention, in order for thecancer-specific tumor antigen epitope to be loaded on theantigen-presenting cell, such loading may be performed using a fusionprotein that contains the cancer-specific tumor antigen epitope providedin the present invention; and a dendritic cell-specific antibody or afragment thereof.

According to still yet another embodiment of the present invention,there is provided a T cell activated by an antigen-presenting cellprovided in the present invention.

In the present invention, the T cells refer to a population ofmonoclonal (for example, encoding the same TCR) or polyclonal (forexample, having clones encoding different TCRs) T cells that have T cellreceptors recognizing a tumor antigen peptide, and may include one ormore subtypes of T cells, including, but not limited to, one or moreselected from the group consisting of cytotoxic T cells, helper T cells,natural killer T cells, γδ T cells, regulatory T cells, and memory Tcells, with memory T cells being preferred.

In the present invention, the “memory T cells” are T cells that havepreviously encountered and responded to their specific antigen, or Tcells that have differentiated from activated T cells. Althoughtumor-specific memory T cells make up a small portion of the total Tcell amount, they play an important function in surveillance of tumorcells during a person's entire lifespan. In a case where tumor-specificmemory T cells encounter tumor cells that express their specific tumorantigen, the memory T cells are immediately activated and clonallyexpanded. The activated and expanded T cells differentiate into effectorT cells to kill tumor cells with high efficiency. Memory T cells areimportant for establishing and maintaining long-term tumorantigen-specific responses of T cells. In the present invention,activated T cells, preferably activated memory T cells, specificallyrecognize antigens on cancer cells, so that such T cells can treat acancerous or neoplastic condition or prevent recurrence, progression, ormetastasis of cancer while avoiding the defense mechanism of cancercells.

According to still yet another embodiment of the present invention,there is provided a method for activating T cells using anantigen-presenting cell (APC) provided in the present invention.

In the present invention, for activation of the T cells, the T cells maybe co-cultured with antigen-presenting cells loaded with acancer-specific tumor antigen epitope of the present invention.

In the present invention, the T cells may be obtained from varioussources including autologous sources, that is, derived from a targetindividual, may preferably be obtained from peripheral blood mononuclearcells (PBMCs) derived from peripheral blood, and may more preferably beobtained from non-adherent portions of the peripheral blood mononuclearcells. In an example of the present invention, the non-adherent portionsof the PBMCs may be obtained by density gradient centrifugation of aperipheral blood sample, or may be obtained by performing culture withat least one cytokine (such as IL-2) in the presence or absence of ananti-CD3 antibody (such as OKT3).

In the present invention, the T cells refer to a population ofmonoclonal (for example, encoding the same TCR) or polyclonal (forexample, having clones encoding different TCRs) T cells that have T cellreceptors recognizing a tumor antigen peptide, and may include one ormore subtypes of T cells, including, but not limited to, one or moreselected from the group consisting of cytotoxic T cells, helper T cells,natural killer T cells, γδ T cells, regulatory T cells, and memory Tcells, with memory T cells being preferred.

In addition, in the present invention, the T cells and theantigen-presenting cells may be derived from the same individual, suchas an individual suffering from cancer, preferably EBV-positive cancer(for example, low to medium grade cancer). However, the presentinvention is not limited thereto.

In the present invention, for activation of the T cells, the T cells maybe co-cultured with antigen-presenting cells of the present inventionfor any one or more time periods of 1, 2, 3, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, or 30 days, and preferably for 1 to 21 days, 1to 14 days, 2 to 10 days, 2 to 5 days, 2 to 5 days, 3 days, 5 days, 7days, 10 days, 14 days, 16 days, 18 days, or 21 days. However, thepresent invention is not limited thereto.

In the present invention, during the co-culture of the T cells withantigen-presenting cells of the present invention, one or more cytokinesmay be added to prime the T cells so that activation, maturation and/orproliferation of the T cells are promoted and the T cells subsequentlydifferentiate into memory T cells. Exemplary cytokines that may be usedat this stage include, but are not limited to, interleukin-2 (IL-2),interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-15 (IL-15),interleukin-21 (IL-21), or combinations thereof, and the like.

In addition, in the present invention, during the co-culture of the Tcells with antigen-presenting cells of the present invention, a fusionprotein comprising a cytokine and an immunoglobulin heavy chain constantregion may be added to prime the T cells so that activation, maturationand/or proliferation of the T cells are promoted and the T cellssubsequently differentiate into memory T cells. Here, the cytokine mayinclude, but is not limited to, interferon-γ (IFN-γ), interleukin-2(IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), IL-18, and tumornecrosis factor (TNF), or granulocyte macrophage colony stimulatingfactor (GMCSF). The immunoglobulin heavy chain constant region may alsobe, but is not limited to, an immunoglobulin hinge region, and animmunoglobulin heavy chain constant region optionally selected from thegroup consisting of CH2 domain, CH3 domain, and CH4 domain, orcombinations thereof. In addition, the immunoglobulin heavy chainconstant region may be derived from immunoglobulins belonging to any offive immunoglobulin classes called in the art as IgA (Igα), IgD (Igδ),IgE (Igϵ), IgG (Igγ), and IgM (Igμ), and may preferably be animmunoglobulin heavy chain constant region derived from the IgG class.

In addition, in the present invention, during the co-culture of the Tcells with antigen-presenting cells of the present invention, a fusionprotein that contains ligand binding to a cell surface protein which ishighly expressed in memory T cells; and an immunoglobulin heavy chainconstant region, may be added to prime the T cells so that activation,maturation and/or proliferation of the T cells are promoted and the Tcells subsequently differentiate into memory T cells. Here, the cellsurface protein which is highly expressed in memory T cells may be CD27,CXCR3, or CD62L. The ligand capable of binding to CD27 may be CD70; theligand capable of binding to CXCR3 may be CXCR9 or CXCR10; and theligand capable of binding to CD62L may be GlyCAM-1, CD34, MadCAM-1, orPSGL-1. However, the present invention is not limited thereto. Inaddition, the immunoglobulin heavy chain constant region may be derivedfrom immunoglobulins belonging to any of five immunoglobulin classescalled in the art as IgA (Igα), IgD (Igδ), IgE (Igϵ), IgG (Igγ), and IgM(Igμ), and may preferably be an immunoglobulin heavy chain constantregion derived from the IgG class.

According to still yet another embodiment of the present invention,there is provided an immunotherapeutic agent, comprising, as an activeingredient, an antigen-presenting cell loaded with a cancer-specifictumor antigen epitope provided in the present invention. Theimmunotherapeutic agent according to the present invention can increaseimmune responses or may selectively increase some of immune responsesdesired for treatment or prevention of a certain disease, for example,cancer.

According to still yet another embodiment of the present invention,there is provided an anticancer vaccine or a pharmaceutical compositionfor preventing or treating cancer, comprising, as an active ingredient,an antigen-presenting cell loaded with a cancer-specific tumor antigenepitope provided in the present invention; and/or an activated T cell.

As used herein, the term “cancer” refers to or indicates a physiologicalcondition characterized by cell growth in mammals which is not regulatedin a typical manner. The cancer to be prevented, ameliorated, or treatedin the present invention may be Epstein-Barr virus (EBV)-positivecancer, including, but not limited to, EBV-positive gastric cancer,EBV-positive cervical cancer, EBV-positive Burkitt's lymphoma,EBV-positive T cell lymphoma, EBV-positive breast cancer, EBV-positiveleiomyosarcoma, EBV-positive smooth muscle tumor, EBV-positive Hodgkinlymphoma, EBV-positive nasopharyngeal cancer, or EBV-positivepost-transplant lymphoproliferative disorder (PTLD), with EBV-positivegastric cancer being preferred.

The antigen-presenting cell provided in the present invention enablesinduction of differentiation and proliferation of EBV-positive cancerantigen-specific T cells, preferably memory T cells, and the memory Tcells thus activated can treat a cancerous or neoplastic condition orprevent recurrence, progression, or metastasis of cancer while avoidingthe defense mechanism of cancer cells.

The anticancer vaccine according to the present invention may involveboth an immunization method performed by single administration and animmunization method performed by continuous administration. In thepresent invention, the “prevention” may include, without limitation, anyact of blocking symptoms of cancer, or suppressing or delaying thesymptoms, using the pharmaceutical composition of the present invention.

In addition, in the present invention, the “treatment” may include,without limitation, any act of ameliorating or beneficially alteringsymptoms of cancer, using the pharmaceutical composition of the presentinvention.

In the present invention, the pharmaceutical composition may becharacterized by being in the form of capsules, tablets, granules,injections, ointments, powders, or beverages, and the pharmaceuticalcomposition may be characterized by being targeted to humans.

In the present invention, the pharmaceutical composition may beformulated in the form of oral preparations such as powders, granules,capsules, tablets, and aqueous suspensions, preparations for externaluse, suppositories, and sterile injectable solutions, respectively,according to conventional methods, and used. However, the pharmaceuticalcomposition is not limited thereto. The pharmaceutical composition ofthe present invention may further comprise a pharmaceutically acceptablecarrier. As the pharmaceutically acceptable carrier, a binder, aglidant, a disintegrant, an excipient, a solubilizer, a dispersant, astabilizer, a suspending agent, a pigment, a flavor, and the like may beused for oral administration; a buffer, a preserving agent, apain-relieving agent, a solubilizer, an isotonic agent, a stabilizer,and the like may be used in admixture for injections; and a base, anexcipient, a lubricant, a preserving agent, and the like may be used fortopical administration. The preparations of the pharmaceuticalcomposition of the present invention may be prepared in various ways bybeing mixed with the pharmaceutically acceptable carrier as describedabove. For example, for oral administration, the pharmaceuticalcomposition may be formulated in the form of tablets, troches, capsules,elixirs, suspensions, syrups, wafers, or the like. For injections, thepharmaceutical composition may be formulated in the form of unit dosageampoules or multiple dosage forms. Alternatively, the pharmaceuticalcomposition may be formulated into solutions, suspensions, tablets,capsules, sustained-release preparations, or the like.

Meanwhile, as examples of carriers, diluents, or excipients suitable formaking preparations, lactose, dextrose, sucrose, sorbitol, mannitol,xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin,calcium phosphate, calcium silicate, cellulose, methyl cellulose,microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,mineral oil, or the like may be used. In addition, a filler, ananti-coagulant, a lubricant, a wetting agent, a fragrance, anemulsifier, a preservative, and the like may further be included.

The route of administration of the pharmaceutical composition of thepresent invention includes, but is not limited to, oral, intravenous,intramuscular, intraarterial, intramedullary, intradural, intracardiac,transdermal, subcutaneous, intraperitoneal, intranasal, intestinal,topical, sublingual, or rectal route. Oral or parenteral administrationis preferred.

As used herein, the term “parenteral” includes subcutaneous,intradermal, intravenous, intramuscular, intraarticular, intrabursal,intrasternal, intradural, intralesional, and intracranial injection orinfusion techniques. The pharmaceutical composition of the presentinvention may also be administered in the form of suppositories forrectal administration.

The pharmaceutical composition of the present invention may varydepending on a variety of factors, including activity of a certaincompound used, the patient's age, body weight, general health status,sex, diet, time of administration, route of administration, rate ofexcretion, drug combination, and severity of a certain disease to beprevented or treated. A dose of the pharmaceutical composition may varydepending on the patient's condition, body weight, severity of disease,drug form, route of administration, and duration, and may beappropriately selected by those skilled in the art. The pharmaceuticalcomposition may be administered in an amount of 0.0001 to 50 mg/kg or0.001 to 50 mg/kg, per day. Administration may be made once a day orseveral times a day. The dose is not intended to limit the scope of thepresent invention in any way. The pharmaceutical composition accordingto the present invention may be formulated in the form of pills,sugar-coated tablets, capsules, liquids, gels, syrups, slurries, orsuspensions.

According to still yet another embodiment of the present invention,there is provided a method for preventing or treating cancer, comprisinga step of administering, to a target individual, an antigen-presentingcell loaded with a cancer-specific tumor antigen epitope provided in thepresent invention; and/or an activated T cell.

In the present invention, the cancer may be Epstein-Barr virus(EBV)-positive cancer, including, but not limited to, EBV-positivegastric cancer, EBV-positive cervical cancer, EBV-positive Burkitt'slymphoma, EBV-positive T cell lymphoma, EBV-positive breast cancer,EBV-positive leiomyosarcoma, EBV-positive smooth muscle tumor,EBV-positive Hodgkin lymphoma, EBV-positive nasopharyngeal cancer, orEBV-positive post-transplant lymphoproliferative disorder (PTLD), withEBV-positive gastric cancer being preferred.

Dose, schedule, and route of administration of the antigen-presentingcell loaded with a cancer-specific tumor antigen epitope provided in thepresent invention or the activated T cell may be determined depending onthe size and condition of an individual, and in accordance with standardpharmaceutical practice. Exemplary routes of administration includeintravenous, intraarterial, intraperitoneal, intrapulmonary,intravascular, intramuscular, intratracheal, subcutaneous, intraocular,intrathecal, or transdermal route.

A dose of cells administered to an individual may vary depending, forexample, on the particular type of cells being administered, the routeof administration, and the particular type and stage of cancer beingtreated. The amount should be sufficient to produce a desirableresponse, such as a therapeutic response against cancer, but withoutsevere toxicity or adverse events. In some embodiments, the amount ofactivated T cells or antigen-presenting cells (such as dendritic cells)to be administered is a therapeutically effective amount. In someembodiments, the amount of cells (such as dendritic cells loaded with acancer-specific tumor antigen epitope or activated T cells) is an amountsufficient to decrease the size of a tumor, decrease the number ofcancer cells, or decrease the growth rate of a tumor by any one of atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%,as compared with the corresponding tumor size, number of cancer cells,or tumor growth rate in the same individual prior to treatment or ascompared with the corresponding activity in other individuals having notreceived the treatment. The magnitude of effects may be measured usingstandard methods, such as in vitro assays with purified enzymes,cell-based assays, animal models, or experiments using humans.

In an embodiment of the present invention, the antigen-presenting cells(such as dendritic cells) loaded with a cancer-specific tumor antigenepitope of the present invention may be administrated at a dose of anyof 1×10⁵ to 5×10⁵, 5×10⁵ to 1×10⁶, 1×10⁶ to 2×10⁶, 2×10⁶ to 3×10⁶, 3×10⁶to 4×10⁶, 4×10⁶ to 5×10⁶, 5×10⁶ to 6×10⁶, 6×10⁶ to 7×10⁶, 7×10⁶ to8×10⁶, 8×10⁶ to 1×10⁸, 1×10⁶ to 3×10⁶, 3×10⁶ to 5×10⁶, 5×10⁶ to 7×10⁶,2×10⁶ to 4×10⁶, 1×10⁶ to 5×10⁶, or 5×10⁶ to 1×10⁷ cells/individual.However, the present invention is not limited thereto.

In another embodiment of the present invention, the antigen-presentingcells (e.g., dendritic cells) loaded with a cancer-specific tumorantigen epitope of the present invention may be administrated at a doseof any of 1×10⁴ to 5×10⁴, 5×10⁴ to 1×10⁵, 1×10⁵ to 2×10⁵, 2×10⁵ to4×10⁵, 4×10⁵ to 6×10⁵, 6×10⁵ to 8×10⁵, 8×10⁵ to 1×10⁶, 1×10⁶ to 2×10⁶,2×10⁶ to 1×10⁷, 1×10⁴ to 1×10⁵, 1×10⁵ to 1×10⁶, 1×10⁶ to 1×10, 1×10⁴ to1×10⁶, or 1×10⁵ to 1×10⁷ cells/kg. However, the present invention is notlimited thereto.

In addition, in an embodiment of the present invention, the activated Tcells of the present invention may be administrated at a dose of any of1×10⁸ to 5×10⁸, 5×10⁸ to 9×10⁸, 9×10⁸ to 1×10⁹, 1×10⁹ to 2×10⁹, 2×10⁹ to3×10⁹, 3×10⁹ to 4×10⁹, 4×10⁹ to 5×10⁹, 5×10⁹ to 6×10⁹, 6×10⁹ to 1×10¹⁰,1×10⁹ to 3×10⁹, 3×10⁹ to 5×10⁹, 5×10⁹ to 7×10⁹, 7×10⁹ to 1×10¹⁰, 1×10⁹to 5×10⁹, 5×10⁹ to 1×10¹⁰, 3×10⁹ to 7×10⁹, 1×10¹° to 1.5×10¹⁰, 1×10¹⁰ to2×10¹⁰, or 1×10⁹ to 1×10¹⁰ cells/individual. However, the presentinvention is not limited thereto.

In another embodiment of the present invention, the activated T cells ofthe present invention may be administrated at a dose of any of 1×10⁷ to1 ×10⁸, 1×10⁸ to 2×10⁸, 2×10⁸ to 4×10⁸, 4×10⁸ to 6×10⁸, 6×10⁸ to 8×10⁸,8×10⁸ to 1×10⁹, 1×10⁹ to 2×10⁹, 2×10⁹ to 4×10⁹, 4×10⁹ to 1×10¹⁰, 2×10⁸to 6×10⁸, 6×10⁸ to 1×10⁹, 1×10⁸ to 2×10⁸, 2×10⁸ to 2×10⁹, 1×10⁷ to1×10⁸, 1×10⁸ to 1×10⁹, 1×10⁹ to 1×10¹⁰, or 1×10⁷ to 1×10⁹ cells/kg.However, the present invention is not limited thereto.

In the present invention, a stabilizer or excipient such as humanalbumin may be used together with administration of theantigen-presenting cells (such as dendritic cells) loaded with acancer-specific tumor antigen epitope and/or the activated T cells.

In the present invention, dose and dosing schedule of theantigen-presenting cells (such as dendritic cells) loaded with acancer-specific tumor antigen epitope and/or the activated T cells maybe adjusted over the course of treatment based on the judgment of theadministering physician. In some embodiments, the activated T cells maybe administered at any time point of about 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, or 1 month after the antigen-presenting cells loaded with atumor antigen peptide are administered, or may be administeredsimultaneously with the antigen-presenting cells. However, the presentinvention is not limited thereto.

In the present invention, administration of the antigen-presenting cells(such as dendritic cells) loaded with a cancer-specific tumor antigenepitope and/or the activated T cell may be done alone or in combinationwith other therapies, such as surgery, radiation therapy, gene therapy,immunotherapy, bone marrow transplantation, stem cell transplantation,hormone therapy, targeted therapy, cryotherapy, ultrasound therapy,photodynamic therapy, chemotherapy, or the like. Additionally, a personhaving a greater risk of developing a proliferative disease may receivetreatments to inhibit and/or delay development of the disease.

Advantageous Effects of Invention

The antigen-presenting cell, that is, dendritic cell, loaded with anEpstein-Barr virus (EBV)-positive cancer-specific tumor antigen epitopeprovided in the present invention enables rapid and effective inductionof differentiation and proliferation of cancer antigen-specific T cells,preferably memory T cells, and the memory T cells thus activated cantreat an Epstein-Barr virus (EBV)-positive cancerous or neoplasticcondition or prevent recurrence, progression, or metastasis of cancerwhile avoiding the defense mechanism of cancer cells.

In the conventional adoptive T cell therapies, it takes a long time of 3to 6 months to produce a large number of T cells for treatment of cancerpatients, which poses a big problem in the cell production process inimmune cell therapy. However, according to the present invention, 10⁹autologous memory T cells, which should be used for patient treatment,can be produced within three weeks, and cost reduction and minimizedinfection risk factors to external contaminants can be achieved.Accordingly, according to the present invention, there is provided atechnique that can be applied to terminal cancer patients because such atechnique makes rapid therapeutic approaches available for a largernumber of solid cancer patients.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates results obtained by performing ELISPOT on respectiveEBNA-1-derived neoepitopes to identify degree of T cell activationcaused by such neoepitopes in SNU719 cell line according to anembodiment of the present invention.

FIG. 2 illustrates results obtained by performing ELISPOT on respectiveEBNA-1-derived neoepitopes to identify degree of T cell activationcaused by such neoepitopes in EBV-infected MKN74 cell line according toan embodiment of the present invention.

FIG. 3 illustrates results obtained by performing ELISPOT on respectiveLMP-2A-derived neoepitopes to identify degree of T cell activationcaused by such neoepitopes in SNU719 cell line according to anembodiment of the present invention.

FIG. 4 illustrates results obtained by performing ELISPOT on respectiveLMP-2A-derived neoepitopes to identify degree of T cell activationcaused by such neoepitopes in EBV-infected MKN74 cell line according toan embodiment of the present invention.

FIG. 5 illustrates results obtained by performing Cr51 release assay oneach of three neoepitopes, which have been selected from EBNA-1-derivedneoepitopes, in SNU-719 cell line, depending on HLA type, to identifytargeted cancer cell-killing activity of EBV-positive gastric cancercell antigen, caused by human-derived memory T cells produced accordingto an embodiment of the present invention.

FIG. 6 illustrates results obtained by performing Cr51 release assay oneach of three neoepitopes, which have been selected from EBNA-1-derivedneoepitopes, in EBV-infected MKN74 cell line, depending on HLA type, toidentify targeted cancer cell-killing activity of EBV-positive gastriccancer cell antigen, caused by human-derived memory T cells producedaccording to an embodiment of the present invention.

FIG. 7 illustrates results obtained by performing Cr51 release assay oneach of three neoepitopes, which have been selected from LMP-2A-derivedneoepitopes, in SNU-719 cell line, depending on HLA type, to identifytargeted cancer cell-killing activity of EBV-positive gastric cancercell antigen, caused by human-derived memory T cells produced accordingto an embodiment of the present invention.

FIG. 8 illustrates results obtained by performing Cr51 release assay oneach of three neoepitopes, which have been selected from LMP-2A-derivedneoepitopes, in EBV-infected MKN74 cell line, depending on HLA type, toidentify targeted cancer cell-killing activity of EBV-positive gastriccancer cell antigen, caused by human-derived memory T cells producedaccording to an embodiment of the present invention.

FIG. 9 illustrates results obtained by performing Cr51 release assay oneach of three selected EBNA-1-derived neoepitopes in EBV-negative MKN74cell line, to identify cancer cell specificity of such neoepitopes amongEBV-positive gastric cancer cell antigens, caused by human-derivedmemory T cells produced according to an embodiment of the presentinvention.

FIG. 10 illustrates results obtained by performing Cr51 release assay oneach of three selected EBNA-1-derived neoepitopes in EBV-negative MKN74cell line, to identify cancer cell specificity of such neoepitopes amongEBV-positive gastric cancer cell antigens, caused by human-derivedmemory T cells produced according to an embodiment of the presentinvention.

FIG. 11 illustrates results obtained by performing Cr51 release assay oneach of three selected LMP-2A-derived neoepitopes in EBV-negative MKN74cell line, to identify cancer cell specificity of such neoepitopes amongEBV-positive gastric cancer cell antigens, caused by human-derivedmemory T cells produced according to an embodiment of the presentinvention.

FIG. 12 illustrates results obtained by performing Cr51 release assay oneach of three selected LMP-2A-derived neoepitopes in EBV-negative MKN74cell line, to identify cancer cell specificity of such neoepitopes amongEBV-positive gastric cancer cell antigens, caused by human-derivedmemory T cells produced according to an embodiment of the presentinvention.

FIG. 13 illustrates experimental results on effects ofneoepitope-specific cytotoxic T cells in BALB/c nude mouse xenograftmodel (SNU-719), to verify an in vivo anticancer effect ofEBNA-1-derived neoepitopes according to an embodiment of the presentinvention.

FIG. 14 illustrates experimental results on effects ofneoepitope-specific cytotoxic T cells in BALB/c nude mouse xenograftmodel (EBV-infected MKN74), to verify an in vivo anticancer effect ofEBNA-1-derived neoepitopes according to an embodiment of the presentinvention.

FIG. 15 illustrates experimental results on effects ofneoepitope-specific cytotoxic T cells in BALB/c nude mouse xenograftmodel (SNU-719), to verify an in vivo anticancer effect ofLMP-2A-derived neoepitopes according to an embodiment of the presentinvention.

FIG. 16 illustrates experimental results on effects ofneoepitope-specific cytotoxic T cells in BALB/c nude mouse xenograftmodel (EBV-infected MKN74), to verify an in vivo anticancer effect ofLMP-2A-derived neoepitopes according to an embodiment of the presentinvention.

DETAILED DESCRIPTION OF INVENTION

According to an embodiment of the present invention, there is providedan Epstein-Barr virus (EBV)-positive cancer-specific tumor antigenneoepitope, represented by any one of SEQ ID NOs: 1 to 184.

According to another embodiment of the present invention, there isprovided an antigen-presenting cell (APC) loaded with a cancer-specifictumor antigen neoepitope provided in the present invention.

According to yet another embodiment of the present invention, there isprovided a T cell activated by an antigen-presenting cell provided inthe present invention.

According to still yet another embodiment of the present invention,there is provided an anticancer vaccine or a pharmaceutical compositionfor preventing or treating cancer, comprising, as an active ingredient,an antigen-presenting cell loaded with a cancer-specific tumor antigenepitope provided in the present invention; and/or an activated T cell.

Hereinafter, the present invention will be described in more detail byway of examples. These examples are only for describing the presentinvention in more detail, and it will be apparent to those skilled inthe art that according to the gist of the present invention, the scopeof the present invention is not limited by these examples.

EXAMPLES Example 1 Production Method of Autologous Memory T CellsSpecific to EBV-Positive Gastric Cancer Cells and Clinical ApplicationThereof

1. Selection of EBV-Positive Gastric Cancer Cell Antigen Neoepitopes

Algorithms for predicting the most important sequence with accumulationof genetic mutations in gastric cancer cells and for predicting epitopesof this sequence which bind to HLA of T cells were developed usingbioinformatics and proteomics. As algorithms for predicting neoepitopes,NetMHC and NetCTLpan were used. Here, peptide sequences were identifiedwhich are expected to have high binding affinity with various HLA typesincluding HLA-A*1101, HLA-A*0206, HLA-A*3101, HLA-B*5101, HLA-B*4403,HLA-B*5401, HLA-B*5801, and HLA-B*3501, as well as HLA-A*2402,HLA-A*A0201, and HLA-A*3303 which are HLA types that Koreans express themost. To this end, existing EBV+gastric cancer cell lines were HLA-typedto investigate the HLA type of each cell line, and neoepitopes havinghigh binding affinity with each HLA type were predicted and identifiedthrough NetMHC program for the representative proteins, LMP-2A andEBNA-1 proteins, which are present only in EBV+gastric cancer cells dueto EBV virus and cause malignancies. The results are shown in Tables 1and 2, respectively.

TABLE 1 Neoepitopes for LMP-2A protein HLA- HLA- HLA- HLA- HLA- HLA-# ID SEQ (9mer) A*24:02 A*02:01 A*33:03 A*11:01 A*02:06 A*31:01LMP2A_SNU- MGS 27371.8 11991.5 19548.9 21044.7 2968.1 20300.8 719_1 LEMVPM (SEQ ID NO: 1) LMP2A_SNU- SMN 9889.5 9.9 6933.4 4394.9 14.9 1649.6719_119 PVCL PV (SEQ ID NO: 2) LMP2A_SNU- NPV 40322.3 27033.9 31415.938783.6 19110.2 38909.2 719_121 CLPV IV (SEQ ID NO: 3) LMP2A_SNU- CLPV13941.3 10007.3 11545.2 4017.4 5530.2 10510.1 719_124 IVAP Y (SEQ ID NO:4) LMP2A_SNU- LPVI 29593.2 19280.3 23285.5 37445.1 8770.7 30098.9719_125 VAP YL (SEQ ID NO: 5) LMP2A_SNU- VIVA 3787.8 21911.8 24575.210829.4 13529.2 16840.6 719_127 PYLF W (SEQ ID NO: 6) LMP2A_SNU- IVAP2732.2 10.1 5448.5 8930.6 6.7 2015.8 719_128 YLF WL (SEQ ID NO: 7)LMP2A_SNU- VAP 6485.2 2405.6 10912 20624.5 159.1 10250.1 719_129 YLF WLA(SEQ ID NO: 8) LMP2A_SNU- APY 39372.1 14186.6 22554 23424.5 5249.720785.8 719_130 LFW LAA (SEQ ID NO: 9) LMP2A_SNU- PYLF 45.8 20451.820179 37721.6 12845 12993.1 719_131 WLA AI (SEQ ID NO: 10) LMP2A_SNU-YLF 36946.9 22.5 13714.8 22720.8 67.5 15292.2 719_132 WLA AIA (SEQ IDNO: 11) LMP2A_SNU- LAAI 11127 28133.2 32080.5 26643.1 12921.5 32823.8719_136 AAS CF (SEQ ID NO: 12) LMP2A_SNU- AIAA 30319.9 200.5 24584.26731.1 71.3 12568.3 719_138 SCFT A (SEQ ID NO: 13) LMP2A_SNU- AAS25117.4 224.6 17740.8 12369.6 32.8 11110 719_140 CFTA SV (SEQ ID NO: 14)LMP2A_SNU- FTAS 19464.9 88.6 13762.4 20136.1 14.6 19810.8 719_144 VST VV(SEQ ID NO: 15) LMP2A_SNU- ASVS 37052.1 5560.8 32852.6 8850.6 424.718831.4 719_146 TVV TA (SEQ ID NO: 16) LMP2A_SNU- TATG 28638.1 14742.631449.6 28040.2 4055.3 34638 719_153 LALS L (SEQ ID NO: 17) LMP2A_SNU-GLA 18374.7 83 33237 18495.4 254.8 19863.2 719_156 LSLL LL (SEQ ID NO:18) LMP2A_SNU- LALS 35190.6 817.2 21235.6 14830.1 83.6 15856.8 719_157LLLL A (SEQ ID NO: 19) LMP2A_SNU- ALSL 40341.5 157.1 31542.6 14530.5 19017991.5 719_158 LLLA A (SEQ ID NO: 20) LMP2A_SNU- LSLL 25009.2 295.114717.7 21696.2 30.5 9125.3 719_159 LLA AV (SEQ ID NO: 21) LMP2A_SNU-SLLL 38650.3 362.5 30858 23970.9 591.4 22057.6 719_160 LAA VA (SEQ IDNO: 22) LMP2A_SNU- LLLA 39044.6 402.5 31171.1 21818.6 315.9 23555.9719_162 AVAS S (SEQ ID NO: 23) LMP2A_SNU- LLA 26232.6 11237.2 17769.41693.5 8474.3 15035 719_163 AVAS SY (SEQ ID NO: 24) LMP2A_SNU- AAV38779.4 7069.2 29746.7 10683.7 397.4 19865.3 719_165 ASSY AA (SEQ ID NO:25) LMP2A_SNU- AVAS 39351.2 1556.8 21477.3 6403.6 124.8 14250.3 719_166SYA AA (SEQ ID NO: 26) LMP2A_SNU- ASSY 40984.7 33783 367.6 173.6 25710.576.6 719_168 AAA QR (SEQ ID NO: 27) LMP2A_SNU- SSYA 36886.5 31558.35931.4 14.5 20818 930.3 719_169 AAQ RK (SEQ ID NO: 28) LMP2A_SNU- AQR23509.8 882.2 24447.6 21770.7 70 5273.4 719_174 KLLT PV (SEQ ID NO: 29)LMP2A_SNU- KLLT 11776.9 93.5 28522.5 24228.3 193.5 7097.1 719_177 PVT VL(SEQ ID NO: 30) LMP2A_SNU- LLTP 37137.6 147.1 23494.6 22140.1 257.420809.9 719_178 VTV LT (SEQ ID NO: 31) LMP2A_SNU- TPVT 37734.2 18144.224195.8 32539.5 5162.9 31765.6 719_180 VLTA V (SEQ ID NO: 32) LMP2A_SNU-TVLT 1299.4 12607.8 21331.9 12630.8 2596 17811.8 719_183 AVV TF (SEQ IDNO: 33) LMP2A_SNU- VLTA 1880 1724.7 18858.8 13899.4 1269.2 11444.8719_184 VVT FF (SEQ ID NO: 34) LMP2A_SNU- LTAV 28599.4 522 4795.7 3078.192.6 3678 719_185 VTFF A (SEQ ID NO: 35) LMP2A_SNU- TAV 7615.8 3348.312820.8 15877.6 134.9 13801.6 719_186 VTFF AI (SEQ ID NO: 36) LMP2A_SNU-TFFA 209.6 24870.2 17421.4 26303.1 22323.9 16909.8 719_190 ICLT W (SEQID NO: 37) LMP2A_SNU- FFAI 31407.7 22080.5 67.9 9575.9 20572.3 140.7719_191 CLT WR (SEQ ID NO: 38) LMP2A_SNU- FAIC 2489.3 33.1 6603.320325.8 8.7 11143.4 719_192 LTW RI (SEQ ID NO: 39) LMP2A_SNU- SLLF33552.8 28.8 22637.9 7154.3 26.2 12754.2 719_207 ALL AA (SEQ ID NO: 40)LMP2A_SNU- LLFA 39864.6 17.1 15143.5 19287.8 22.5 11438.6 719_208 LLA AA(SEQ ID NO: 41) LMP2A_SNU- ALL 30890.7 238.2 33730 31528.9 425.5 22061.4719_211 AAA GGL (SEQ ID NO: 42) LMP2A_SNU- AAA 27565 3517.1 32696.624960 137.2 27509.3 719_214 GGL QGI (SEQ ID NO: 43) LMP2A_SNU- GLQ19757.3 26.7 18903.5 20338.6 89.9 6778.5 719_218 GIYV LV (SEQ ID NO: 44)LMP2A_SNU- LQGI 21973.1 8022.8 37703.6 30905.4 2345.8 29019 719_219 YVLVM (SEQ ID NO: 45) LMP2A_SNU- GIYV 36065.3 151.8 21377.9 13972.3 448.88123.9 719_221 LVM LV (SEQ ID NO: 46) LMP2A_SNU- YVL 17490.5 293.6 1515627530.7 153.6 11887.4 719_223 VML VLL (SEQ ID NO: 47) LMP2A_SNU- VML35525.8 444.6 24613.5 18421.9 674.5 10636.3 719_226 VLLI LA (SEQ ID NO:48) LMP2A_SNU- MLV 37081.4 19470 23627.1 6144.4 16629.6 20171.2 719_227LLIL AY (SEQ ID NO: 49) LMP2A_SNU- LVLL 40296.1 15526.5 27.5 405.910119.1 20.7 719_228 ILAY R (SEQ ID NO: 50) LMP2A_SNU- VLLI 33407.115983.6 238.7 964.6 16825.3 563 719_229 LAY RR (SEQ ID NO: 51)LMP2A_SNU- LLIL 37733.4 194672 353 3782.7 15867.3 159.2 719_230 AYR RR(SEQ ID NO: 52) LMP2A_SNU- LILA 10191.2 25457.8 25429.9 27362.6 19540.717519.7 719_231 YRR RW (SEQ ID NO: 53) LMP2A_SNU- ILAY 39845.2 28729.171.9 7319.8 30463.6 29.4 719_232 RRR WR (SEQ ID NO: 54) LMP2A_SNU- LAY24998.6 20425.9 7.8 424.1 14678.8 5.2 719_233 RRR WRR (SEQ ID NO: 55)LMP2A_SNU- RLTV 30154.3 7330.9 345477 24745.1 5573.4 16855.2 719_241CGGI M (SEQ ID NO: 56) LMP2A_SNU- LTVC 9930 15172.7 21640.9 14515.6 382420535.8 719_242 GGI MF (SEQ ID NO: 57) LMP2A_SNU- TVC 17100.9 384.89819.9 8431.6 128.5 9625.5 719_243 GGI MFL (SEQ ID NO: 58) LMP2A_SNU-GGI 29382.6 2824.6 25826.7 29467 437.1 15686.2 719_246 MFL ACV (SEQ IDNO: 59) LMP2A_SNU- IMFL 13468.2 33.7 7969.8 17351.1 93.2 2903 719_248ACV LV (SEQ ID NO: 60) LMP2A_SNU- FLAC 19384.4 10.5 12112.4 24245.6 16.810846 719_250 VLV LI (SEQ ID NO: 61) LMP2A_SNU- VLV 31115.8 63.7 27382.529178.3 81.1 22651.6 719_254 LIVD AV (SEQ ID NO: 62) LMP2A_SNU- LIVD23873.3 143.2 25164.7 21696.4 59.9 25241.6 719_257 AVL QL (SEQ ID NO:63) LMP2A_SNU- AVL 10808.3 395.5 23133.1 13646.3 117.1 9438.6 719_261QLSP LL (SEQ ID NO: 64) LMP2A_SNU- LQLS 32042.4 469.3 29672.4 14340.629.8 15058.9 719_263 PLLG A (SEQ ID NO: 65) LMP2A_SNU- QLSP 33550.6 49.721128.2 26797.4 43.7 16755 719_264 LLG AV (SEQ ID NO: 66) LMP2A_SNU-SPLL 30117.5 17918.9 30290.4 33256.4 7730 31488.4 719_266 GAV TV (SEQ IDNO: 67) LMP2A_SNU- VTV 15006.2 5526.8 18969.7 15331.5 589.6 13347.5719_272 VSM TLL (SEQ ID NO: 68) LMP2A_SNU- TVV 6049.7 652.7 13530.97958.2 177.7 15315.7 719_273 SMT LLL (SEQ ID NO: 69) LMP2A_SNU- VVS8120.1 978.1 21960 10661 428 13853.2 719_274 MTL LLL (SEQ ID NO: 70)LMP2A_SNU- VSM 22765.4 920 16803.5 2487.7 79.4 5629.8 719_275 TLLL LA(SEQ ID NO: 71) LMP2A_SNU- SMT 2867.4 5041.4 25983.4 15082.4 3004.416672.7 719_276 LLLL AF (SEQ ID NO: 72) LMP2A_SNU- MTL 19035.5 27.41099.5 4770.9 8.4 582 719_277 LLLA FV (SEQ ID NO: 73) LMP2A_SNU- TLLL12676.6 164.2 20545 25952.5 587.5 13178.3 719_278 LAF VL (SEQ ID NO: 74)LMP2A_SNU- LLLA 24803 40 23132.4 26986.3 202.3 10568 719_280 FVL WL (SEQID NO: 75) LMP2A_SNU- LLAF 3324 300. 1301 1175 412. 9140 719_281 VLW LS(SEQ ID NO: 76) LMP2A_SNU- TLG 7510.8 95.9 31160 23852.4 183.4 22528.4719_296 AAL LTL (SEQ ID NO: 77) LMP2A_SNU- AAL 39908.7 1586.9 28364.316855.7 86.9 16521.3 719_299 LTLA AA (SEQ ID NO: 78) LMP2A_SNU- ALLT23261.1 46.8 26860.1 27729.8 155.2 13582.5 719_300 LAA AL (SEQ ID NO:79) LMP2A_SNU- LTLA 11578.4 949 20215.5 12875.3 129.5 13315.3 719_302AAL AL (SEQ ID NO: 80) LMP2A_SNU- TLA 6696.7 21.8 13358.6 16222.7 28.211363.2 719_303 AAL ALL (SEQ ID NO: 81) LMP2A_SNU- LAA 36062.2 2985.825515.9 15003.8 414 25095.9 719_304 ALA LLA (SEQ ID NO: 82) LMP2A_SNU-AAL 16882.4 797.7 23744.5 21892.9 49.1 10793.2 719_306 ALL ASL (SEQ IDNO: 83) LMP2A_SNU- ALA 14588.2 191 30081.3 19870.7 541.2 18083.4 719_307LLAS LI (SEQ ID NO: 84) LMP2A_SNU- LALL 17259.7 7925.7 33454.2 2993.871744.5 29244 719_308 ASLI L (SEQ ID NO: 85) LMP2A_SNU- YPSA 41679.639870.2 37645.8 39333.8 32297.5 42032 719-31 SGSY G (SEQ ID NO: 86)LMP2A_SNU- LLAS 41604.9 74.7 29015.5 23428.3 99.3 24439.7 719_310 LILG T(SEQ ID NO: 87) LMP2A_SNU- SLIL 12080.2 70.6 25306.7 15799.3 170.616385.8 719_313 GTL NL (SEQ ID NO: 88) LMP2A_SNU- LILG 41776.7 1274.627409.7 27342.2 220.3 24829.1 719_314 TLNL T (SEQ ID NO: 89) LMP2A_SNU-GTL 5555.5 13923.8 23808.1 6308.3 2682.7 9656.2 719_317 NLTT MF (SEQ IDNO: 90) LMP2A_SNU- TLNL 11744.1 70.1 13411.5 9317.2 331.4 9132.5 719_318TTM FL (SEQ ID NO: 91) LMP2A_SNU- NLTT 13538.2 261.2 12244.1 16237.1454.7 15909.4 719_320 MFL LM (SEQ ID NO: 92) LMP2A_SNU- TTM 2651.6 248.11781.8 2219 48.5 1513.6 719_322 FLL MLL (SEQ ID NO: 93) LMP2A_SNU- TMF3988.3 16232.3 24882.1 18622.5 13501.9 17977.3 719_323 LLM LLW (SEQ IDNO: 94) LMP2A_SNU- FLL 8083.8 8.9 12745.7 33192.8 13.9 7882.1 719_325MLL WTL (SEQ ID NO: 95) LMP2A_SNU- LLM 13488.2 26.7 7799.5 17571.3 52.83052 719_326 LLW TLV (SEQ ID NO: 96) LMP2A_SNU- LML 15787.8 31.7 15849.619663.7 119.1 6864 719_327 LWT LVV (SEQ ID NO: 97) LMP2A_SNU- MLL 1691224.7 13754 24171.7 52.5 7678.7 719_328 WTL VVL (SEQ ID NO: 98)LMP2A_SNU- LLW 22368.6 24.7 11762 26130.3 141.6 4070.5 719_329 TLV VLL(SEQ ID NO: 99) LMP2A_SNU- CPLT 37670.6 21444.6 26652.3 29420.8 10764.329121.2 719_345 KILL A (SEQ ID NO: 100) LMP2A_SNU- ILLA 5233.8 3865.66694.9 236 2901.5 1264.9 719_350 RLFL Y (SEQ ID NO: 101) LMP2A_SNU- LLA31672.9 34.9 11717.7 4604.7 45.8 6011.2 719_351 RLFL YA (SEQ ID NO: 102)LMP2A_SNU- RLFL 6856.4 20.3 14775.5 9547.6 92.6 2096.5 719_354 YAL AL(SEQ ID NO: 103) LMP2A_SNU- FLYA 11428.5 6 10051.6 14849.5 19.6 7883.2719_356 LALL L (SEQ ID NO: 104) LMP2A_SNU- LYAL 101 12176 20911.631716.4 6586.9 16497.4 719_357 ALLL L (SEQ ID NO: 105) LMP2A_SNU- YAL31846.1 159.2 16022.1 14850.3 20.8 15655.3 719_358 ALLL LA (SEQ ID NO:106) LMP2A_SNU- LALL 40372 1722.3 23602.9 27980.2 127.2 20356.9 719_360LLAS A (SEQ ID NO: 107) LMP2A_SNU- ALLL 25296.6 167 29618.5 30183.4455.9 16547.6 719_361 LAS AL (SEQ ID NO: 108) LMP2A_SNU- LLLL 7740.755.9 20129.3 23602.9 116.9 13531.6 719_362 ASA LI (SEQ ID NO: 109)LMP2A_SNU- LLLA 37708.1 263.9 35188.7 16754.1 400.9 26159.2 719_363 SALIA (SEQ ID NO: 110) LMP2A_SNU- ALIA 25800.8 493.1 36320.6 28980.7 560.124630.6 719_368 GGSI L (SEQ ID NO: 111) LMP2A_SNU- GSIL 40801.5 35501.66602.4 16.2 26128.9 791.4 719_373 QTN FK (SEQ ID NO: 112) LMP2A_SNU-KSLS 5507.7 2533.5 21307.9 12976.3 221.3 3775.2 719_381 STEF I (SEQ IDNO: 113) LMP2A_SNU- SSTE 14988.3 2681.8 19581.3 13178 219.9 11100.4719_384 FIPN L (SEQ ID NO: 114) LMP2A_SNU- FIPN 4505.5 72.9 1067527973.3 19.8 14050.6 719_388 LFC ML (SEQ ID NO: 115) LMP2A_SNU- IPNL16916.4 10587.1 20005.8 27848 6583.9 20799.9 719_389 FCM LL (SEQ ID NO:116) LMP2A_SNU- NLFC 14595.1 20.1 7777.2 14671.3 74.9 7191.9 719_391 MLLLI (SEQ ID NO: 117) LMP2A_SNU- MLL 22235.9 20.4 9301.5 26251 43.9 6024.6719_395 LIVA GI (SEQ ID NO: 118) LMP2A_SNU- LLIV 6873.4 7266.6 29271.621315.3 4210.1 22774.5 719_397 AGIL F (SEQ ID NO: 119) LMP2A_SNU- LIVA23654.3 75.6 18461.8 15122.1 47.5 14693.4 719_398 GILFI (SEQ ID NO: 120)LMP2A_SNU- IVAG 6760.8 65.7 10025.3 14721.2 34.3 6870.5 719_399 ILFIL(SEQ ID NO: 121) LMP2A_SNU- FILAI 5075.9 3363.4 19730 23123.9 1176.521302.8 719_405 LTE W (SEQ ID NO: 122) LMP2A_SNU- LTE 45205.3 34231.1404.4 1968.7 28770.8 787.8 719_410 WGS GNR (SEQ ID NO: 123) LMP2A_SNU-RTY 13674.3 2452.5 20810.3 7194.6 359.6 2242.3 719_418 GPVF MC (SEQ IDNO: 124) LMP2A_SNU- TYG 43.1 20573.4 17372.1 28767 11909.3 10962.4719_419 PVF MCL (SEQ ID NO: 125) LMP2A_SNU- FMC 36749.1 418.4 26159.727774.5 543.7 23960.5 719_424 LGG LLT (SEQ ID NO: 126) LMP2A_SNU- MCL25037.6 3987 27163.8 30133.1 848.4 26216.1 719_425 GGL LTM (SEQ ID NO:127) LMP2A_SNU- CLG 29360.1 39.8 26562.2 29588.7 70 18962.1 719_426 GLLTMV (SEQ ID NO: 128) LMP2A_SNU- GLLT 42669.3 50.4 33575.3 27874.2 101.919703.9 719_429 MVA GA (SEQ ID NO: 129) LMP2A_SNU- LLT 37594.5 307.427810.6 35011.8 519 26238 719_430 MVA GAV (SEQ ID NO: 130) LMP2A_SNU-LTM 2451.6 22223.6 21501.8 15181.9 8332.6 17938.6 719_431 VAG AVW (SEQID NO: 131) LMP2A_SNU- TMV 14694.7 81.7 20692.6 26012.6 145.8 18008.3719_432 AGA VWL (SEQ ID NO: 132) LMP2A_SNU- MVA 21800.9 136.2 5179.17819.4 24.4 7866.6 719_433 GAV WLT (SEQ ID NO: 133) LMP2A_SNU- VAG8593.3 2606.6 25834.3 23105.9 411.4 17830.3 719_434 AVW LTV (SEQ ID NO:134) LMP2A_SNU- WLT 11823.3 106 26126.6 35724.7 248.7 26604.7 719_439VMT NTL (SEQ ID NO: 135) LMP2A_SNU- LTV 17661.7 3493.6 16154 19986.5509.9 14004.8 719_440 MTN TLL (SEQ ID NO: 136) LMP2A_SNU- VMT 31556.9156.8 28830.3 13858.3 115.4 18258.8 719_442 NTLL SA (SEQ ID NO: 137)LMP2A_SNU- MTN 7290.9 20237.4 12898.6 10001.4 8034.8 12238.2 719_443TLLS AW (SEQ ID NO: 138) LMP2A_SNU- TLLS 30273 396.9 21252 15570.9 352.714095.9 719_446 AWIL T (SEQ ID NO: 139) LMP2A_SNU- LLSA 32764.6 32.424319.4 13902.6 93.4 16585.4 719_447 WILT A (SEQ ID NO: 140) LMP2A_SNU-SAWI 4573.1 11740.2 13701 17240.5 2700.5 8892.1 719_449 LTAG F (SEQ IDNO: 141) LMP2A_SNU- WILT 5519.8 57.6 10388.7 17443.7 33.4 8331.9 719_451AGF LI (SEQ ID NO: 142) LMP2A_SNU- ILTA 769.6 2963.6 29225.1 16621.51785.3 21390.8 719_452 GFLI F (SEQ ID NO: 143) LMP2A_SNU- LTAG 16982.4120 6508.1 8045.4 33.2 4517.1 719_453 FLIF L (SEQ ID NO: 144) LMP2A_SNU-FLIF 42365.3 42.7 13437.3 25165.8 52 16269.2 719_457 LIGF A (SEQ ID NO:145) LMP2A_SNU- IFLIG 43.6 17773.3 13570.3 28036.3 10285.2 10021.4719-459 FALF (SEQ ID NO: 146) LMP2A_SNU- FLIG 36214.7 277.8 25993.627631.3 461.1 25977.5 719_460 FALF G (SEQ ID NO: 147) LMP2A_SNU- LIGF24962.4 20.3 11149.9 18296.8 16.6 6672.9 719_461 ALF GV (SEQ ID NO: 148)LMP2A_SNU- GFAL 35328.8 33229.1 582.4 8108.8 32269.6 210 719_463 FGVI R(SEQ ID NO: 149) LMP2A_SNU- LFG 28785.7 34993.2 78.8 12351.4 33974.738.3 719_466 VIRC CR (SEQ ID NO: 150) LMP2A_SNU- VPM 44669 36655.435264.6 36414.6 26600.4 39954.4 719_7 GAG PPS (SEQ ID NO: 151) HLA- HLA-HLA- HLA- HLA- HLA- # ID B*51:01 B*15:01 B*44:03 B*54:01 B*58:01 B*35:01LMP2A_SNU- 19068 1393.9 31463.2 20902.1 2627.5 164.2 719_1 LMP2A_SNU-18869.2 443.8 11789.2 12880.3 9730 9998.1 719_119 LMP2A_SNU- 1717.434671.7 29524.1 241.1 30432.6 868.3 719_121 LMP2A_SNU- 32273.1 161.217839.2 29638.7 17168.1 1118.9 719_124 LMP2A_SNU- 456.5 22503.1 30741.7337.4 11162.4 39 719_125 LMP2A_SNU- 23369.9 7172.4 14029.8 36881.8 8114816.8 719_127 LMP2A_SNU- 14093.9 7650 29346.4 18362.2 817.2 9877.3719_128 LMP2A_SNU- 21984.5 20546.3 34583.3 6597 10554.1 13623 719_129LMP2A_SNU- 9931.9 20808.5 14633.9 25.3 33807.9 1458.6 719_130 LMP2A_SNU-32690.2 33027.6 35911.5 39974.3 31558.3 38018.7 719_131 LMP2A_SNU-26173.6 3914.4 28938.1 5371.4 30695.2 11886.3 719_132 LMP2A_SNU- 7996.663.4 14661.8 18015.7 159.8 23.8 719_136 LMP2A_SNU- 37957.8 6013.531247.4 14492.5 17396.7 21943.8 719_138 LMP2A_SNU- 9860.7 3306.5 10812.25602.1 3334.9 7527.4 719_140 LMP2A_SNU- 5884.4 1074.2 32054.2 1077.51464.4 1864.9 719_144 LMP2A_SNU- 32015 3547.8 26008.7 9458.8 3367.218947.7 719_146 LMP2A_SNU- 12835.5 9454.7 26436.6 21854.8 1695.6 404.4719_153 LMP2A_SNU- 38515.9 4023.7 31753.5 41085.5 15854.4 36429.6719_156 LMP2A_SNU- 9880.7 9979.6 25269 909.2 2060 6184.2 719_157LMP2A_SNU- 41561.2 6626.9 26400.6 22040.9 30091.4 31931.3 719_158LMP2A_SNU- 7137.4 4766.5 30342.5 3013.5 2491.7 14007.7 719_159LMP2A_SNU- 35597.7 6048.7 26564.8 16328.5 32493.8 27442.7 719_160LMP2A_SNU- 34547.8 3633.5 37258.4 16083.4 27538.7 18507 719_162LMP2A_SNU- 23097.6 9.1 9592.6 23151.9 2410.6 43 719_163 LMP2A_SNU-30204.3 3134.7 19736.8 2929 8128.6 2163.8 719_165 LMP2A_SNU- 36692.71626.1 28170.4 6365.9 23419.2 10828.7 719_166 LMP2A_SNU- 45002.3 24905.234733.7 39245.8 16532.8 34429.5 719_168 LMP2A_SNU- 35890.1 17324.825977.8 33151.5 7653 29593.5 719_169 LMP2A_SNU- 29998.4 223.9 14359.920829.2 28749.6 32884.3 719_174 LMP2A_SNU- 26627.8 787.1 31556.2 34200.46615.5 25782.9 719_177 LMP2A_SNU- 35201.7 13541.2 40609.9 19821.120510.8 21753.5 719_178 LMP2A_SNU- 1073 27088.7 32545.2 36.9 30156.9322.4 719_180 LMP2A_SNU- 17799.8 242.9 18087.5 26212.1 1633.7 303.3719_183 LMP2A_SNU- 28882.1 119.1 16654.5 35748.6 1881.7 4251.7 719_184LMP2A_SNU- 26352.3 14096.2 34361.4 2279.9 2239.6 11683.7 719_185LMP2A_SNU- 1811.7 4697.9 20385.3 1210.6 1509.2 1494.9 719_186 LMP2A_SNU-23353.2 17916.9 7418.5 35167.8 1245.1 14681.3 719_190 LMP2A_SNU- 46034.139399.8 40223.4 35558.1 43345.9 36240.1 719_191 LMP2A_SNU- 701.1 7610.211525.1 679.6 323.5 1097.9 719_192 LMP2A_SNU- 36465.9 3779.6 28678.811933.9 27135 21409.4 719_207 LMP2A_SNU- 25677.4 1228.6 30349.7 280928533 12044.2 719_208 LMP2A_SNU- 35326.5 2099.8 24292.3 40247.3 22863.430329.1 719_211 LMP2A_SNU- 18595.5 8783.3 20531.6 26576 4561.5 20074.7719_214 LMP2A_SNU- 39230.9 15981.5 30612.2 35452.4 29224.7 42802.1719_218 LMP2A_SNU- 28065.4 128 14998.1 33933.9 18243.6 8771 719_219LMP2A_SNU- 35575.8 16510.1 32974.8 28738.1 24709.3 42830.8 719_221LMP2A_SNU- 25310.5 10525.9 35050.4 21845.1 9208.3 13460.3 719_223LMP2A_SNU- 38789 14580.5 34150.1 26232 28571.3 36984.1 719_226LMP2A_SNU- 34382.6 171.6 11119.5 26821.8 11758.2 366.9 719_227LMP2A_SNU- 42621.4 33896.1 40627.1 26214.4 30730.7 34800.3 719_228LMP2A_SNU- 45282.7 36046.9 36738 40449.4 37005.7 40947.5 719_229LMP2A_SNU- 45312.6 23309 39296.3 40381.7 37662.9 38250.9 719_230LMP2A_SNU- 21706.8 9545.3 17745.4 34120.6 136 18865.5 719_231 LMP2A_SNU-46423.7 30236.3 42353.4 42222 40589.7 40637.6 719_232 LMP2A_SNU- 33940.228971 35978.8 22147.5 14714.4 25705.5 719_233 LMP2A_SNU- 34894.5 216.931598.6 33239.9 10678.4 12768 719_241 LMP2A_SNU- 20266.8 151.3 2358721495.2 512.1 692.3 719_242 LMP2A_SNU- 28314.3 5527.2 35112.3 30373.49552.1 13224.3 719_243 LMP2A_SNU- 32710.4 17841.9 32488.9 27472.721090.2 38076.7 719_246 LMP2A_SNU- 19631.8 4985.6 28744 20012.3 20098.427284.3 719_248 LMP2A_SNU- 23637.6 7804.8 28164.6 13581.5 18267.519826.9 719_250 LMP2A_SNU- 25904.3 6865.1 31934.1 18772.4 26216.427905.3 719_254 LMP2A_SNU- 17736.2 1148.6 32569.5 22865.1 6475.4 3550.7719_257 LMP2A_SNU- 20883.6 4239.7 18649.1 30555 4025.1 11797.6 719_261LMP2A_SNU- 28135.4 2373.8 17703.8 14681.8 21815.1 27746.3 719_263LMP2A_SNU- 26585.2 3605.7 30283.5 19801.6 25397.5 24460.1 719_264LMP2A_SNU- 1526.9 29720.3 28761.4 355 27198.2 892.6 719_266 LMP2A_SNU-21080.7 3762.7 36890.5 30202.3 452.5 18717.1 719_272 LMP2A_SNU- 15908.71878.8 25562.4 21519.7 2484.5 3325.5 719_273 LMP2A_SNU- 19204.7 2901.832228.4 28795 1653.4 11146 719_274 LMP2A_SNU- 15701.6 6979.6 21951.23615.2 826.9 18865.5 719_275 LMP2A_SNU- 33160.8 60 8048.1 35866 7268.31565.1 719_276 LMP2A_SNU- 11617.1 11509.4 27039.5 2957.3 2041.4 20396.6719_277 LMP2A_SNU- 31870.9 9836 27846.8 32047.2 21002.6 19043.3 719_278LMP2A_SNU- 32574.4 13364.1 29749.2 34396.7 14753.9 32068.7 719_280LMP2A_SNU- 4290 2287 3489 2588 2376 3206 719_281 LMP2A_SNU- 30173.31555.3 33269.7 37369 12634.3 20706.8 719_296 LMP2A_SNU- 23912.1 4561.123387.8 2285.5 13858.1 8526 719_299 LMP2A_SNU- 30515.3 753.6 16063.932101.7 21011.9 14789.4 719_300 LMP2A_SNU- 8078.8 561.8 27998.1 10984.6188.7 641.6 719_302 LMP2A_SNU- 26107.4 612.3 25912.1 30321.5 1064712400.7 719_303 LMP2A_SNU- 9761.3 5008 22831.3 743 798.5 1496.1 719_304LMP2A_SNU- 13608.2 1354.4 14455.4 17031.9 928.5 3403.9 719_306LMP2A_SNU- 25036 3146.9 16547.8 32603.7 12979.9 35350.6 719_307LMP2A_SNU- 3865.7 3163.4 29938.1 12036 436.8 600.4 719_308 LMP2A_SNU-13480.7 25105.7 38370.8 653.1 25946.6 290.6 719-31 LMP2A_SNU- 38239.810950.6 35332.6 27090.7 26687.8 31919.2 719_310 LMP2A_SNU- 29842.7 677.124228.8 35236.8 15685.2 15131.9 719_313 LMP2A_SNU- 36325.3 20529.843873.4 17673.7 30129.5 28730.9 719_314 LMP2A_SNU- 32438.7 337.5 19058.336479.3 182.7 7933.2 719_317 LMP2A_SNU- 26916.6 1882.1 25585.3 3078612223.1 10896.2 719_318 LMP2A_SNU- 27240.3 2283.2 19381.3 29707.815477.7 3864.1 719_320 LMP2A_SNU- 18366.4 3871.8 21160.2 13386.7 625.411838.4 719_322 LMP2A_SNU- 28013.9 7739 5484.2 39287 377.9 19971.6719_323 LMP2A_SNU- 29633 9838.9 29389.6 24180.3 18410.8 15012.5 719_325LMP2A_SNU- 22552.3 6274.9 22518.5 11468.4 21324.5 27980.5 719_326LMP2A_SNU- 15437.3 2864 29283.3 15663.8 15936 17141 719_327 LMP2A_SNU-20823.4 1237.6 27968.7 15626.1 11511.6 5123.8 719_328 LMP2A_SNU- 26938.76287.6 30004.3 24819.7 16723.5 22476.8 719_329 LMP2A_SNU- 3795.9 33986.832242.7 40.5 27618.4 4556.3 719_345 LMP2A_SNU- 28103.1 675.1 15314.731828.5 3987.8 3393.9 719_350 LMP2A_SNU- 32842.3 4290.9 32551.5 9100.620431 25441.8 719_351 LMP2A_SNU- 22423.6 352.6 17945 23931.5 8333.211218.5 719_354 LMP2A_SNU- 14452.3 933.8 22566.5 14066.6 8976 7114.4719_356 LMP2A_SNU- 27210.3 16018.8 33860.9 34946.7 16177.4 23396.4719_357 LMP2A_SNU- 11348.3 9221.5 22015.4 558.3 2741.6 2807.8 719_358LMP2A_SNU- 12404.7 7468.9 31337.1 726.9 8043.6 5539.5 719_360 LMP2A_SNU-30580.1 982.2 18787.5 32238.5 24767.1 17945.6 719_361 LMP2A_SNU- 10169.72337.2 27104.5 20273.6 10813.2 22288.7 719_362 LMP2A_SNU- 28071.2 3395.534432.5 11384 20770 18889.4 719_363 LMP2A_SNU- 32742.3 102.5 21901.834824 20077.8 14272.5 719_368 LMP2A_SNU- 43973.3 24934.3 39868.5 40321.414126.7 38027.7 719_373 LMP2A_SNU- 18540.5 8780.4 27824.2 27091 48.429173.2 719_381 LMP2A_SNU- 18233.9 11500.3 25381.8 32592.1 521 12130.1719_384 LMP2A_SNU- 18845.9 3217.7 37974.7 17265.7 27948.8 8245.1 719_388LMP2A_SNU- 808.3 22119.5 23895.3 1101.4 14068.7 298.1 719_389 LMP2A_SNU-17987 10340.4 16613.5 17723.3 17321.2 28299.3 719_391 LMP2A_SNU- 16888.95920 19812.8 13208.7 14228.6 26095.6 719_395 LMP2A_SNU- 28410.7 114.519338.1 30961 7162.9 4153.9 719_397 LMP2A_SNU- 15242 4774.9 34571.417750.6 7809.7 20384.2 719_398 LMP2A_SNU- 20780.6 3961.8 33101 18325.31341.3 6022.3 719_399 LMP2A_SNU- 14795.5 4255.1 11400.4 18955.3 741361.9 719_405 LMP2A_SNU- 43444.1 32402.9 44760 36137.2 34919.5 29665719_410 LMP2A_SNU- 33440.4 22294 35295.9 20698.7 1940.2 38397.7 719_418LMP2A_SNU- 31268.4 22881.4 30885.4 36459.2 27367.3 23665 719_419LMP2A_SNU- 38175.3 6987.3 39059 27342.8 32303.8 26047.9 719_424LMP2A_SNU- 15294.2 3697.3 26509.3 18716.3 1800.8 227.3 719_425LMP2A_SNU- 33113.9 18368.6 35968.2 31684.9 29085.3 41790.7 719_426LMP2A_SNU- 41913.5 12690.2 31166.7 32767.4 34065.6 41039.7 719_429LMP2A_SNU- 18858.1 2762.7 34930.4 14473.4 28951.9 17382.8 719_430LMP2A_SNU- 5820 331.9 4419.9 14170.2 3.5 575.5 719_431 LMP2A_SNU-30304.4 1675.5 20684.8 36242.1 12071.7 10316.5 719_432 LMP2A_SNU-26470.9 14570.8 28749 4146.1 5216 5224.5 719_433 LMP2A_SNU- 5520.811126.7 28783.8 8098 946.5 14748.6 719_434 LMP2A_SNU- 21999.9 1453.331394.8 22179.2 13818.5 2582.2 719_439 LMP2A_SNU- 14075.1 1202 34902.118094.4 339 6061.1 719_440 LMP2A_SNU- 30682.5 1108.2 32059.4 17305.716675.9 18845.3 719_442 LMP2A_SNU- 12564 770.3 3529.2 16440 4.4 383.6719_443 LMP2A_SNU- 41198.1 22402.5 35064.1 28311.3 32570.2 33655.3719_446 LMP2A_SNU- 29325.5 3688.2 31123.2 12418.8 16408.9 19392.4719_447 LMP2A_SNU- 10008.4 210.4 4177.6 15491.4 309.7 149.2 719_449LMP2A_SNU- 9748.3 10058.9 24364.2 8275.5 7233.1 10872.3 719_451LMP2A_SNU- 21963.6 41.1 20551.4 32421.1 3330.1 1209.6 719_452 LMP2A_SNU-24710.9 6108.3 33208.9 19951.9 1093.2 12051.5 719_453 LMP2A_SNU- 37931.19963.8 38572.6 13793.5 39126.6 29860.5 719_457 LMP2A_SNU- 27325.6 5467.119946.5 32621 14748.5 7339.5 719-459 LMP2A_SNU- 36790.5 11054.1 32861.925757.5 25267.6 26034.4 719_460 LMP2A_SNU- 30790.6 17419.9 38901.620134.8 18600.8 33095.9 719_461 LMP2A_SNU- 48129.4 37637.2 36865.842648.6 44000.4 40388.2 719_463 LMP2A_SNU- 46970.9 39068.3 42482.339408.7 43194.2 37968.9 719_466 LMP2A_SNU- 17537.7 28276.4 40505.1 191.338119.6 337.5 719_7

TABLE 2 Neoepitopes for EBNA-1 protein SEQ HLA- HLA- HLA- HLA- HLA- HLA-# ID (9mer) A*24:02 A*02:01 A*33:03 A*11:01 A*02:06 A*31:01 EBNA- RGR45316.5 39680 4373 18740.2 34826.3 289.3 1_SNU- GGS 719_277 GGR (SEQ IDNO: 152) EBNA- RGR 45316.5 39680 4373 18740.2 34826.3 289.3 1_SNU- GGS719_285 GGR (SEQ ID NO: 153) EBNA- RGR 45316.5 39680 4373 18740.234826.3 289.3 1_SNU- GGS 719_293 GGR (SEQ ID NO: 154) EBNA- RGR 46032.142644 3895.5 29673.7 38658.7 229.8 1_SNU- GRER 719_302 AR (SEQ ID NO:155) EBNA- RAR 45029.1 39602.4 1219 10439.7 31841.9 86.1 1_SNU- GGSR719_308 ER (SEQ ID NO: 156) EBNA- GSRE 47184.8 41384.4 1249.1 15361.737002.5 175.9 1_SNU- RAR 719_312 GR (SEQ ID NO: 157) EBNA- RAR 44722.239536 884.7 17784.2 33601.8 65.1 1_SNU- GRG 719_316 RGR (SEQ ID NO: 158)EBNA- RGR 44053.2 42854.4 3516.3 22983.4 38461.4 136.3 1_SNU- GRG719_320 EKR (SEQ ID NO: 159) EBNA- RGR 45689.7 42293.4 4657.9 28956.638106 233.2 1_SNU- GEK 719_322 RPR (SEQ ID NO: 160) EBNA- SSSS 42342.436614.5 425.2 125.9 28232.3 177.9 1_SNU- GSPP 719_336 R (SEQ ID NO: 161)EBNA- HPV 31231.2 36176.7 33692.8 36983.6 22741.8 41710.8 1_SNU- GDA719_355 DYF (SEQ ID NO: 162) EBNA- KGG 42456.6 37922.1 4856.2 1012033694.3 124.7 1_SNU- WFG 719_409 KHR (SEQ ID NO: 163) EBNA- RGR 45006.242700.3 3668.5 27677.6 39018.4 188.3 1_SNU- GRG 719_41 RGR (SEQ ID NO:164) EBNA- EGLR 42050.2 37488.5 187.6 7955.5 29022.1 987.3 1_SNU- VLLA719_431 R (SEQ ID NO: 165) EBNA- RVLL 19512.6 2581.5 11516.2 16095.2353.6 1075 1_SNU- ARSH 719_434 V (SEQ ID NO: 166) EBNA- LLAR 36376.118739.8 116.8 577.6 17709.7 53 1_SNU- SHVE 719_436 R (SEQ ID NO: 167)EBNA- RGR 45332.2 41211.9 3488.8 26198.8 37068.6 236.3 1_SNU- GRG 719_45GGR (SEQ ID NO: 168) EBNA- GVF 44626.5 27517.3 9143.4 81.9 20473.51986.2 1_SNU- VYG 719_454 GSK (SEQ ID NO: 169) EBNA- KTSL 34794.626595.5 446.4 53.6 20319.7 18.8 1_SNU- YNL 719_462 RR (SEQ ID NO: 170)EBNA- IALA 39511.7 32880.4 527.2 2884.4 26713.5 229.8 1_SNU- VPQC719_472 R (SEQ ID NO: 171) EBNA- ITPLS 1491.1 25301 24504 16803.110821.1 17909.2 1_SNU- RLPF 719_481 (SEQ ID No: 172) EBNA- RESI 13257.417730.8 23507 22586.8 4054.7 14022.8 1_SNU- VCYF 719_503 M (SEQ ID NO:173) EBNA- ESIV 17345.4 5738.8 5835.7 18953.5 465.9 9913.9 1_SNU- CYF719_504 MV (SEQ ID NO: 174) EBNA- SIVC 2026.1 6030.4 15283.4 5288.31655.6 10406.6 1_SNU- YFM 719_505 VF (SEQ ID NO: 175) EBNA- IVCY 26724.5521.2 7210.1 10733.8 480.8 3174.7 1_SNU- FMV 719_506 FL (SEQ ID NO: 176)EBNA- FMV 10141.7 16.1 10488.4 28823.4 26.1 10274.1 1_SNU- FLQT 719_510HI (SEQ ID NO: 177) EBNA- MVF 773.1 6982.1 6844.4 6047.9 2152.6 8273.31_SNU- LQTH 719_511 IF (SEQ ID NO: 178) EBNA- FLQT 34284.9 428.3 14139.718590.5 402.8 12823.6 1_SNU- HIFA 719_513 E (SEQ ID NO: 179) EBNA- LQTH17941 61.6 16078.5 25417.3 9.8 12729.3 1_SNU- IFAE 719_514 V (SEQ ID NO:180) EBNA- AIKD 39676.1 22195.5 2666.4 29.4 14992.6 322.1 1_SNU- LVM719_526 TK (SEQ ID NO: 181) EBNA- NIKV 15027.2 26519.7 16872.7 27649.517243.3 20453.6 1_SNU- TVCS 719_540 F (SEQ ID NO: 182) EBNA- TVCS40262.6 2096.8 22454.7 24077.5 380.9 24941.6 1_SNU- FDD 719_544 GV (SEQID NO: 183) EBNA- FPPM 43694.8 29057.6 36446.2 42153.1 18949.4 43126.11_SNU- VEG 719_558 AA (SEQ ID NO: 184) HLA- HLA- HLA- HLA- HLA- HLA-# ID B*51:01 B*15:01 B*44:03 B*54:01 B*58:01 B*35:01 EBNA- 47765.829769.2 45986.8 44280.7 39982.1 43547.6 1_SNU- 719_277 EBNA- 47765.829769.2 45986.8 44280.7 39982.1 43547.6 1_SNU- 719_285 EBNA- 47765.829769.2 45986.8 44280.7 39982.1 43547.6 1_SNU- 719_293 EBNA- 47725.532620.6 45707.5 43198 39720.8 41805.7 1_SNU- 719_302 EBNA- 45468.319127.1 42510.4 37732.6 27999.9 30362.9 1_SNU- 719_308 EBNA- 47577.532963.7 43767.7 45223 37929.5 45253.8 1_SNU- 719_312 EBNA- 45327.323182.2 42450.6 38545.9 32356.3 39243.2 1_SNU- 719_316 EBNA- 47659.936580.1 45330.7 42924.9 37426.4 44343.5 1_SNU- 719_320 EBNA- 48265.633970.3 46416.2 44423.2 39930.2 43241.9 1_SNU- 719_322 EBNA- 43513.723101.1 39692.5 36562.7 27088.4 22741.3 1_SNU- 719_336 EBNA- 12468.913575.1 25926.7 10752.9 9492.1 13.2 1_SNU- 719_355 EBNA- 48473.3 35691.144147.2 45552.5 34497.4 44034.7 1_SNU- 719_409 EBNA- 47327.5 33030.845445.2 44144.4 39900.9 44790.1 1_SNU- 719_41 EBNA- 43250.4 39180.940538 35094.5 36902.5 29032.2 1_SNU- 719_431 EBNA- 22726.3 5093.332701.6 14056.7 7788.7 35120.3 1_SNU- 719_434 EBNA- 45126.7 22087.240498.5 35766.4 32575.8 26860.1 1_SNU- 719_436 EBNA- 47258.9 32002.945779.3 43911.9 41157.1 44732.4 1_SNU- 719_45 EBNA- 41747.3 17980.836473 31707.9 30633.8 30968.7 1_SNU- 719_454 EBNA- 45634.8 29342.938073.8 40031.5 13160.8 39576.3 1_SNU- 719_462 EBNA- 37853.6 31599.637944.3 32340.2 17579.5 24121.8 1_SNU- 719_472 EBNA- 26114.5 438.135685.7 33692.8 3771.2 2754 1_SNU- 719_481 EBNA- 33839.3 5262.5 44026568.8 11335.6 16703 1_SNU- 719_503 EBNA- 20573.9 28021.1 28717.39180.2 7673.5 23247.5 1_SNU- 719_504 EBNA- 24843.9 52.8 11265 23057.28440 813.6 1_SNU- 719_505 EBNA- 34592.3 12094.1 39419 32020.9 11647.322473.2 1_SNU- 719_506 EBNA- 10650.8 402 22139.2 16201.6 5493.7 11412.71_SNU- 719_510 EBNA- 4430.3 28.8 10520 7239.1 217.3 20.3 1_SNU- 719_511EBNA- 39689 12298.9 41389.8 23759.7 38950.5 15521.1 1_SNU- 719_513 EBNA-17980.6 1123 14996.1 11684.8 17726.8 17732.5 1_SNU- 719_514 EBNA-44286.5 16408 35567.3 36968.8 30457 32160.8 1_SNU- 719_526 EBNA- 29037.2154.9 24316.2 34414.2 16844.2 1653.5 1_SNU- 719_540 EBNA- 34793.928022.7 35086.1 24905.8 21011.9 27572.1 1_SNU- 719_544 EBNA- 7576.130308.4 45375.9 48.7 39527.1 418.3 1_SNU- 719_558

As shown in Tables 1 and 2 above, through in silico prediction,neoepitopes were identified which have high binding affinity forHLA-A*2402, HLA-A*A0201, HLA-A*3303, HLA-A*1101, HLA-A*0206, HLA-A*3101,HLA-B*5101, HLA-B*4403, HLA-B*5401, HLA-B*5801, or HLA-B*3501, and theirbinding affinity with respective HLA's was predicted by IC₅₀ (nM)values. Sequences present in actual human gastric cancer cells andrecognizable by T cells in the body were selected based on their bindingaffinity with the HLA's. As shown in Table 1, regarding the neoepitopesfor LMP-2A, in a case of HLA-A*2402, TYGPVFMCL (IC₅₀=43.1 nM), LLWTLVVLL(IC₅₀=22368.6 nM), LTEWGSGNR (IC₅₀=45205.3 nM) were selected in order ofhigh to low binding affinity therefor, and in a case of HLA-A*3101,LAYRRRWRR (IC₅₀=5.2 nM), LTVMTNTLL (IC₅₀=14004.8 nM), YPSASGSYG(IC₅₀=42032 nM) were selected. In addition, as shown in Table 2,regarding the neoepitopes for EBNA-1, in a case of HLA-A*2402, MVFLQTHIF(IC₅₀=773.1 nM), AIKDLVMTK (IC₅₀=39676.1 nM), GSRERARGR (IC₅₀=47184.8nM) were selected in order of high to low binding affinity therefor, andin a case of HLA-A*3101, KTSLYNLRR (IC₅₀=18.8 nM), RGRGGSGGR (IC₅₀=289.3nM), FPPMVEGAA (IC₅₀=43126.1 nM) were selected. The neoepitopes selectedas above were synthesized into peptides for the following experiments.

2. ELISPOT Results for T Cells Activated by Dendritic Cells Loaded withSelected Neoepitope

PBMCs extracted from healthy human blood were separated into monocytesand leukocytes through flow cytometry, and the monocytes were culturedfor 2 days in a culture supplemented with cytokines GM-CSF and IL-4 todifferentiate into dendritic cells. In addition, the leukocytes werecultured with anti-CD3/CD28 antibody for 3 days, and then cultured in aculture supplemented with cytokine IL-2. The neoepitope peptide selectedas above was transferred to the monocyte-differentiated dendritic cellsusing electroporation. Subsequently, culture was performed for 2 days toidentify that the neoepitope has been expressed on the surface of thedendritic cells. Then, the dendritic cells were co-cultured with theleukocytes, which were cultured in a culture supplemented withanti-CD3/CD28 antibody, at a ratio of 1:20 (dendritic cells:leukocytes).In a case of the co-culture, the culture was mixed with a cytokinecocktail containing both cytokine IL-4 that increases theantigen-presenting function of dendritic cells, and cytokines IL-2 andIL-7 that function to help conversion of T cells into memory cells, andculture was performed. After 16 hours, expression levels of IFN-γ in theT cells thus activated were measured with ELISPOT, and the results areillustrated in FIGS. 1 to 4. Here, for a negative control peptide usedin the experiments, the amino acid sequence 9-mer (sequence: GGSRERARG)was employed which is of any EBNA-1 or LMP-2A protein that has not beenextracted through NetMHC in EBNA-1 and LMP-2A.

As a result, it was found that the T-cells cultured with dendritic cellsloaded with a neoepitope peptide of the present invention secrete muchmore IFN-γ than the control regardless of binding affinity of thepeptide for HLA.

From these results, it was found that in the present invention,cytotoxic T lymphocytes (CTLs) can be activated by the dendritic cellsloaded with each of neoepitopes, which have been selected by HLA-A typesof respective cell lines in Tables 1 and 2 above, and the thus activatedT cells have antigen specificity which enables recognition of theneoepitope that is a neoantigen.

3. Cancer Cell Lysis Effect of T Cells Activated by Dendritic CellsLoaded with Selected Neoepitope

In order to select memory T cells to which an antigen had been presentedthrough the dendritic cells after co-culture for 72 hours as in item no.2 above, a magnetic-activated cell sorter (MACS) capable of extracting Tcells secreting cytokine IFN-γ was used to extract EBV antigen-specificmemory T cells. The extracted memory T cells were cultured in a culturesupplemented with cytokines IL-2, IL-7, and IL-15 to maintain theirmemory function and increase the number of cells, in which the culturewas performed until the memory T cells reach the number of cells thatcan be injected into mice.

Such activated T cells were co-cultured with SNU-719 cell line, that is,EBV-positive gastric cancer cells, and with EBV-infected MKN74 cellline, to identify lysis of the cancer cells through Cr51 release assay.The results are illustrated in FIGS. 5 to 8. Also, such activated Tcells were co-cultured with MKN-74 cell line, that is, EBV-negativegastric cancer cells, to identify lysis of the cancer cells through Cr51release assay. The results are illustrated in FIGS. 9 to 12. Here, for anegative control peptide used in the experiments, the amino acidsequence 9-mer (sequence: GGSRERARG) was employed which is of any EBNA-1or LMP-2A protein that has not been extracted through NetMHC in EBNA-1and LMP-2A.

As a result, it was found that the T cells activated by the neoepitopeaccording to the present invention exhibit excellent lytic abilityagainst EBV-positive gastric cancer cells while exhibiting insignificantlytic ability against EBV-negative gastric cancer cells. In other words,it can be seen that the T cells activated by the dendritic cells loadedwith the EBV cancer-specific antigen neoepitope according to the presentinvention specifically lyse EBV-positive cancer cells.

In addition, the T cells activated by the neoepitope according to thepresent invention tended to show an increase in the lytic ability, in amixed-concentration dependent manner, especially in a case of beingco-cultured with EBV-positive gastric cancer cells; and it was foundthat in a case where, among the neoepitopes for respective HLA types, inparticular, a neoepitope with higher binding affinity with HLA is used,the activated T cells exhibit a more increased lytic ability againstEBV-positive gastric cancer cell line.

4. Identification of In Vivo Anticancer Effect of T Cells Activated byDendritic Cells Loaded with Selected Neoepitope

The EBV-positive gastric cancer cell line, SNU-719 (HLA type:HLA-A*2402), or EBV-infected MKN74 (HLA type: HLA-A*3101), in an amountof 1×10⁷ cells, was mixed with Matrigel, and the mixture wastransplanted into the flank of BALB/c nude mice, to produce a xenograftmodel. After 4 weeks had elapsed, in a case where cancer tissue isobserved, the T cells activated in item no. 2 above, in an amount of1×10⁷ cells, were intratumorally injected once a week, and then the sizeof cancer tissue was measured. The results are illustrated in FIGS. 13to 16. Here, for a negative control peptide used in the experiments, theamino acid sequence 9-mer (sequence: GGSRERARG) was employed which is ofany EBNA-1 or LMP-2A protein that has not been extracted through NetMHCin EBNA-1 and LMP-2A.

As a result, it was found that in a case where activated T cells aresubjected to treatment with the neoepitope according to the presentinvention, the size of EBV-positive gastric cancer is remarkablydecreased as compared with the control; and it was found that in a casewhere, among the neoepitopes for respective HLA types, in particular, aneoepitope with higher binding affinity with HLA is used, a higher rateof decrease in size of gastric cancer is exhibited.

As described above, it was found that all neoepitopes selected by theHLA-A type of each cell line exhibit an excellent effect in enhancingactivity of T cells, and that as binding affinity obtained bybioinformatics increases (that is, IC₅₀ value decreases), activity ofthe T cells is enhanced and activity thereof for targeted cancer cellkilling is increased. From these results, it is easily predictable thatdendritic cells together with the neoepitopes shown in Tables 1 and 2can be used to enhance activity of T cells, and this also results in anexcellent targeted cancer therapeutic effect.

Although specific parts of the present invention have been described indetail as above, it is obvious to those skilled in the art that such aspecific description is merely a preferred embodiment, and the scope ofthe present invention is not limited thereto. Therefore, the substantialscope of the present invention will be defined by the appended claimsand equivalents thereof.

INDUSTRIAL APPLICABILITY

The present invention relates to a cancer-specific tumor antigenneoepitope, an antigen-presenting cell loaded with the neoepitope, and amethod for activating T cells for cancer treatment using theantigen-presenting cell.

1.-36. (canceled)
 37. A method for preventing or treating cancer,comprising a step of administering to a target individual anantigen-presenting cell loaded with an Epstein-Barr virus (EBV)-positivecancer-specific tumor antigen neoepitope represented by any one of SEQID NOs: 1 to
 184. 38. The method according to claim 37, wherein theantigen-presenting cell is a dendritic cell, a B cell, or a macrophage.39. The method according to claim 37, wherein the antigen-presentingcell promotes proliferation or differentiation of T cells.
 40. Themethod according to claim 37, wherein the Epstein-Barr virus(EBV)-positive cancer-specific tumor antigen neoepitope exhibits bindingaffinity with at least one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G,β2-microglobulin, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1,HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOBloci.
 41. The method according to claim 37, wherein the Epstein-Barrvirus (EBV)-positive cancer-specific tumor antigen neoepitope exhibitsbinding affinity with at least one of HLA-A*2402, HLA-A*A0201,HLA-A*3303, HLA-A*1101, HLA-A*0206, HLA-A*3101, HLA-B*5101, HLA-B*4403,HLA-B*5401, HLA-B*5801, and HLA-B*3501.
 42. The method according toclaim 37, wherein the cancer is EBV-positive gastric cancer,EBV-positive cervical cancer, EBV-positive Burkitt's lymphoma,EBV-positive T cell lymphoma, EBV-positive breast cancer, EBV-positiveleiomyosarcoma, EBV-positive smooth muscle tumor, EBV-positive Hodgkinlymphoma, EBV-positive nasopharyngeal cancer, or EBV-positivepost-transplant lymphoproliferative disorder (PTLD).
 43. A method forproducing an antigen-presenting cell, the method comprising loading theantigen-presenting cell with an Epstein-Barr virus (EBV)-positivecancer-specific tumor antigen neoepitope represented by any one of SEQID NOs: 1 to
 184. 44. The method according to claim 43, wherein theantigen-presenting cell is a dendritic cell, a B cell, or a macrophage.45. The method according to claim 43, wherein the antigen-presentingcell is obtained from peripheral blood mononuclear cells (PBMCs) derivedfrom peripheral blood of a target individual.
 46. The method accordingto claim 43, wherein the loading is performed by contacting or pulsingthe antigen-presenting cell with the cancer-specific tumor antigenneoepitope, wherein the loading is performed by nucleofection of theantigen-presenting cell with an expression vector into which a nucleicacid molecule encoding the cancer-specific tumor antigen neoepitope isinserted, or wherein the loading is performed using a fusion proteinthat contains the cancer-specific tumor antigen neoepitope and adendritic cell-specific antibody or a fragment thereof.
 47. A method forpreventing or treating Epstein-Barr virus (EBV)-positive cancer,comprising a step of administering to a target individual a T cellactivated by an antigen-presenting cell loaded with an Epstein-Barrvirus (EBV)-positive cancer-specific tumor antigen neoepitoperepresented by any one of SEQ ID NOs: 1 to
 184. 48. A method foractivating T cells, the method comprising contacting the T cells with anantigen-presenting cell loaded with an Epstein-Barr virus (EBV)-positivecancer-specific tumor antigen neoepitope represented by any one of SEQID NOs: 1 to
 184. 49. The method according to claim 48, wherein thecontacting is performed by co-culture of the T cells with theantigen-presenting cell.
 50. The method according to claim 48, whereinthe T cells are obtained from peripheral blood mononuclear cells (PBMCs)of a target individual.
 51. The method according to claim 48, whereinthe T cells include one or more selected from the group consisting ofcytotoxic T cells, helper T cells, natural killer T cells, γδT cells,regulatory T cells, and memory T cells.
 52. The method according toclaim 49, wherein the co-culture is performed with addition ofinterleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7),interleukin-15 (IL-15), interleukin-21 (IL-21), or a combinationthereof.
 53. The method according to claim 49, wherein the co-culture isperformed with addition of a fusion protein that contains a cytokine andan immunoglobulin heavy chain constant region.
 54. The method accordingto claim 53, wherein the cytokine is interferon-γ (IFN-γ), interleukin-2(IL-2), interleukin-4 (IL-4), interleukin-12 (IL-12), IL-18, tumornecrosis factor (TNF), or granulocyte macrophage colony stimulatingfactor (GMCSF).
 55. The method according to claim 49, wherein theco-culture is performed with addition of a fusion protein that contains(a) ligand of CD27, CXCR3, or CD62L and (b) an immunoglobulin heavychain constant region.